Asymmetric synthesis of peptides

ABSTRACT

A process comprising substitution of an acceptor molecule comprising a group —XC(O)— wherein X is O, S, or NR 8 , where R 8  is C 1-6  alkyl, C 6-12  aryl or hydrogen, with a nucleophile, wherein the acceptor molecule is cyclised such that said nucleophilic substitution at —XC(O)— occurs without racemization, is provided. This process has particular application for the production of a peptide by extension from the activated carboxy-terminus of an acyl amino acid residue without epimerization.

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No. 12/066,727, filed Mar. 13, 2008, which is the U.S. national stage of International Application No. PCT/GB05/03797, which designated the United States and was filed on Sep. 30, 2005, published in English, which claims priority under 35 U.S.C. §119 or 365 to United Kingdom, Application No. 0518667.1, filed 13 Sep. 2005. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a novel process for peptide synthesis, by the addition of amino acids to the activated C-terminus of a peptide chain. Peptide synthesis is central to the manufacture of many drugs and medicaments. Peptides or derivatives thereof are used for the treatment of many disorders from antibiotics to anticancer agents. Therefore improving peptide synthesis and the yield of peptide produced by chemical synthesis has been the focus of much research in recent years.

Chemical synthesis of proteins or peptides has been a particular focus in the art. The chemical synthesis of proteins or peptides allows the production of purified peptides of specific amino acid sequence. It also allows the production of truncated sequences of amino acids and allows the introduction of non-natural amino acid derivatives.

Proteins are produced in nature by the stepwise condensation of amino acid monomers on a ribosome. Synthesis of the protein begins from the N-terminal residue and grows towards the C-terminus. The conventional approach to peptide synthesis has concentrated on extension at the N-terminus of a growing peptide. This approach forms the basis of conventional solid phase peptide synthesis. Peptide synthesis solely by extension from the N-terminus is limiting as it renders any peptide synthesis linear in nature, rather than convergent. This can severely increase overall length of synthesis, increase operational time and decrease overall yield with consequent possibilities for the loss of stereochemical fidelity.

To overcome the problems associated with N-terminal extension of a peptide, it could be envisaged that the synthesis could instead provide extension of a peptide from the C-terminal. However, attempts at peptide synthesis in the N to C direction have been generally unsuccessful due to epimerisation of the carboxy-terminal amino acid residue. This is due to the tendency of carboxy-terminal-activated acylamino acids and peptides to form oxazolones. As illustrated below, the formation of the oxazolone allows rapid racemisation of the alpha-position of the terminal amino acid residue of the acyl amino acid or peptide.

This racemisation prevents the production of stereochemically homogeneous peptides by C-terminus extension.

It will be appreciated that the production of isomerically pure compounds is a particular requirement in the art. Any method which results in the production of a mixture of isomers will require the use of time consuming and expensive purification steps to separate the isomers. Chiral compounds which are administered to humans or animals are usually required in enantiomerically pure form. The presence of unwanted isomers even in low concentrations can reduce the potency of the compound and can produce unwanted and in some cases disastrous side effects. The incorporation of an unwanted enantiomer into a peptide chain (for example the incorporation of a D-amino acid into a peptide composed of L-amino acids) may disrupt the folding and/or 3D shape of the peptide, thus resulting in a peptide which may have unpredictable binding activities and/or biological properties. The production of enantiomerically pure peptides is therefore of paramount importance.

Various attempts have been made to overcome this problem. Iorga, B and Campagne, J-M (2004, Synlett 10, 1826-1828) attempted to reduce the degree of epimerisation by improving the rate of peptide bond formation over the rate of oxazolone formation, so that peptide bond formation was the predominant reaction. However, this method does not entirely prevent the formation of the oxazolone and therefore epimerisation at the carboxy-terminal activated amino acid residue occurs. Native chemical ligation has been developed to overcome problems of carboxy-terminal extension but is ordinarily restricted to couplings in which the amino-terminal partner is an assisting cysteine residue and is not applicable to general techniques of automated solid phase peptide synthesis. The application of native chemical ligation to other amino-terminal amino acids has had very limited success.

The present invention provides a new method of producing a peptide by extension from the activated carboxy-terminus of an acyl amino acid residue. This new method overcomes the problems of epimerisation of the terminal amino acid residue during the coupling step. The present invention therefore allows the production of peptides by a convergent approach and provides a new method for the production of potentially biologically important compounds instead of the linear repetitive amino terminal extension approach currently used.

The first aspect of the present invention provides a process comprising substitution of an acceptor molecule comprising a group —XC(X)— (preferably —X(CO)—) wherein each X is independently O, S or NR⁸, where R⁸ is hydrogen, aliphatic group or an aromatic group, preferably hydrogen, C₁₋₆ alkyl, C₆₋₁₂ aryl, with a nucleophile, wherein the acceptor molecule is cyclised such that said nucleophilic substitution at —XC(X)— occurs without racemisation. The acceptor molecule is preferably a cyclised amino acid or derivative thereof. In particular, the acceptor molecule is a compound of formula (II):

wherein each X is O, S, or NR⁸, where R⁸ is as defined above; R² is independently selected from an aliphatic group, such as a C₁₋₁₀ branched or straight chain alkyl group, or an aromatic group, such as C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group, each optionally substituted with a group including, for example, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; R³ is as defined for R² or is hydrogen,

or a group —C(R^(1′)) (R⁹)—N(R¹⁰)(R¹¹); wherein R^(1′) is independently selected from an aliphatic group such as C₁₋₁₀ branched or straight chain alkyl group, an aromatic group, such as C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group, each optionally substituted with a group such as OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R₁₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; wherein when Y is NR⁸, R⁸ and R^(1′) can together form a 4 to 7 membered ring, optionally substituted with a group such as CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more additional heteroatoms selected from O, S or N; R¹² is hydrogen, C₁₋₆ alkyl, C₆₋₁₂ aryl or N(R¹³)₂, wherein each occurrence of R¹³ is independently hydrogen, C₁₋₆ alkyl or C₆₋₁₂ aryl, and R^(4′) is a carboxyl protecting group or hydrogen; R⁹ and R¹⁰ are independently hydrogen or a group as defined for R^(1′); R¹¹ is hydrogen or an amino protecting group preferably selected from a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a 2-nitrophenylsulphenyl group; or R⁹ and R¹⁰ or R¹⁰ and R¹¹ or R^(1′) and R¹⁰ or two R¹³ can together form a 4 to 7 membered ring, optionally substituted with a group such as CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more additional heteroatoms selected from O, S or N; Y is O, S or NR⁸, where R⁸ is as defined above; or YR^(4′) is R³; R⁵ is an aromatic group such as C₆₋₁₂ aryl, C₅₋₁₂ heteroalkyl or an aliphatic group such as C₁₋₈ branched or straight chain alkyl optionally substituted with a group such as OR¹³, SR¹³N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³), or a linker for attachment of formula (II) to a resin or a linked resin; n is 0, 1, 2 or 3 and m is an integer, such as an integer selected from 1-100.

The nucleophilic substitution of the acceptor molecule preferably occurs without epimerisation.

Preferably, the process is carboxy terminal extension of an acceptor molecule, for example an amino acid or peptide. The invention therefore provides a process for the synthesis of a peptide or a peptide analog by carboxy terminal extension, by the addition of a nucleophile to an acceptor molecule, such as a compound of formula (II).

There is further provided a process for the production of a compound of formula (I)

comprising reaction of a compound of formula (II) or (II′) (above) with a compound of formula (III) HY—R⁷  (III) wherein the variables are defined as above; preferably X is O, S, or NR⁸, where R⁸ is as defined above; Y is O, S or NH; R² is independently selected from a C₁₋₁₀ branched or straight chain alkyl group, C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group, optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; R³ is as defined for R² or is hydrogen, or a group

or a group —C(R^(1′))(R⁹)—N(R¹⁰)(R¹¹); wherein R^(1′) is hydrogen or as defined from R¹ below; Y is as defined above and R⁴ is as defined for R⁴ below; R¹² is hydrogen, C₁₋₆ alkyl, C₆₋₁₂ aryl or N(R¹³)₂, wherein each occurrence of R¹³ is independently hydrogen, C₁₋₆ alkyl or C₆₋₁₂ aryl, R⁹ and R¹⁰ are independently hydrogen or a group as defined for R^(1′): or R⁹ and R¹⁰ can together form a 4 to 7 membered ring, optionally substituted with CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more additional heteroatoms selected from O, S or N, R¹¹ is hydrogen or an amino protecting group preferably selected from a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a 2-nitrophenylsulphenyl group; R⁵ is an aromatic group, such as C₅₋₁₂ aryl, C₅₋₁₂ heteroalkyl or an aliphatic group, such as C₁₋₈ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CO N(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂ or a linker for attachment of formula (II) to a resin or a linked resin; R⁶ is hydrogen or

wherein R⁵ and X are as defined above; R⁷ is a chiral, substituted methylene, such as a group

or is independently selected from an aliphatic group such as a C₁₋₁₀ branched or straight chain alkyl group or an aromatic group, such as a C₆₋₁₂ aryl group, optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂, or wherein R⁷ and Y together form a 4 to 7 membered ring, optionally substituted with a group such as OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more heteroatoms in addition to Y, selected from O, S or N; wherein R¹ is R^(1′) or is independently selected from an aliphatic group such as C₁₋₁₀ branched or straight chain alkyl group, or an aromatic group such as C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group optionally substituted with a group such as OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂, and R⁴ is R^(4′) or a carboxyl protecting group or hydrogen; n is 0, 1, 2 or 3 and m is an integer such as a value selected from 1-100 and when n=0, R⁶ is H.

The inventors have surprisingly found that activation of an amino acid or peptide via a cyclic compound as exemplified in formula (II) prevents the formation of an oxazolone thereby allowing the condensation of a compound of formula (III) without concommitant epimerisation. The invention therefore provides peptides via C-terminus extension, said peptides being produced in an enantiomerically and diastereochemically pure form.

The use of activated cyclic N-acyl amino acids, peptides or derivatives thereof eliminates oxazolone formation and associated epimerisation. The use of cyclic activated intermediates in the present invention provides an improved method of peptide synthesis via carboxy-terminal extension.

Therefore, rather than merely reducing the probability of epimerisation occurring, as has been attempted in the prior art, the process of the present invention does not permit epimerisation and therefore guarantees the production of a peptide of correct stereochemistry as the activated carboxyl terminus is held in a cyclic template such that the adjacent amide cannot form the oxazolone.

In accordance with usual practice, * denotes a stereocenter (asymmetric center). Where a compound contains a stereocenter (whether marked in the present application with * or not) the stereochemistry of the asymmetric centers may be in the R or S configuration. The compounds of the present application can be provided in enantiomerically pure form or as a mixture of isomers (including a racemic mixture). Preferably, the compounds of the present inventions are provided in an enantiomerically pure form. The present invention allows maintenance of the desired stereochemistry throughout the synthetic pathway. Thus wherein Y is NH, the amino acids to be attached may be of U or D configuration as required.

Preferably R¹ and R² are independently selected from C₁₋₄ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂, preferably optionally substituted with OH, SH, NH₂, CO₂H, CONH₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NH(C═NH)NH₂.

More preferably, R¹ and R² are independently selected from C₁ alkyl optionally substituted with OH, SH, CO₂H, CONH₂, phenyl, imidazolyl, indolyl or hydroxyphenyl; C₂ alkyl optionally substituted with OH, CO₂H, CONH₂ or SCH₃; C₃ alkyl optionally substituted with NHC(═NH)NH₂; or C₄ alkyl optionally substituted with NH₂.

The integer, m is preferably 1-50, more preferably 1 to 30, most preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The integer n is preferably 0 or 1.

When X is NR, R is preferably Ci₋₄ alkyl, more preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or tert-butyl, phenyl, naphthyl, anthracenyl or phenanthracenyl, more preferably phenyl or hydrogen.

R³ may also be substituted pipecolic acid or derivative thereof, α-alkoxy-α-amino acids, cc, α-diamino acids, β-substituted dehydroamino acids, canavanine, cysteinesulphonamide, homocysteinesulphonamide, γ,δ-unsaturated amino acids. substituted 4-hydroxyprolines, 4-hydroxtyornithines, imino sugars, Fmoc-BPC—OH, Fmoc-TPG-OH and Fmoc-CAA-OH, or (5)-3,5-dihydroxyphenylglycine.

It will be appreciated by a person skilled in the art that amino acids, hydroxy acids and derivatives thereof contain functional groups which require protection. In particular it is known in the art to protect the amino terminus, the carboxyl terminus and/or the side chains of an amino acid or peptide (for example wherein R¹ or R² is C₂CO₂H or CH₂CH₂OH). Examples of such protection are well known in the art. In particular the amino terminus of an amino acid may be protected by one or more of a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a 2-nitrophenylsulphenyl group. The carboxyl group can be protected by one or more of an ester group especially a methyl, ethyl, benzyl, t-butyl or phenyl ester. Thus R⁴ is preferably methyl, ethyl, benzyl, t-butyl or phenyl.

Conditions for the removal of the protecting groups discussed above are well known in the art. The protecting groups may be removed after each coupling reaction (for example, the carboxyl protection) or alternatively at the end of the synthesis (for example, the side chain protection and/or the N-terminal group).

In a particular feature of the first aspect, the invention provides a process for production of a compound of formula (Ia)

comprising reacting a compound of formula (IIa) (above) with a compound of formula (III) HY—R⁷: Wherein the groups Y, X, R², R³, R⁵, and R⁷ are as defined above. In an alternative feature of the first aspect, the invention provides a process for production of a compound of formula (Ib)

comprising reacting a compound of formula (IIb)

with a compound of formula (III) HY—R⁷ wherein the groups Y, X, R², R³, R⁵ and R⁷ are as defined above for compounds (I), (II) and (III).

The N and the terminal ester of formula (I), (Ia) or (Ib) can be unmasked by processes known in the art. for example, sodium liquid ammonia in the presence of an alcohol when R¹=phenyl and R⁴=t-butyl. Alternatively, the ester can be further derivatized, including for example, amidation.

The nucleophilic substitution of the acceptor molecule of the first aspect of the invention can be carried out using reaction conditions known in the art. In some circumstances, for example where the nucleophile and/or the acceptor molecule are sterically hindered it may be necessary for example to use high pressure such as around 19-20 bar, and/or longer reaction times such as 12-72 hours, preferably 24-48 hours. Alternatively, the reaction can be carried out in the presence of a reagent such as AlMe₃. When the substitution is carried out on the solid phase, the reaction can be promoted by the use of an excess of nucleophile.

The invention further relates to a process for the production of a compound of formula (II) (above) by the reaction of a compound of formula (IV)

with a compound of formula (V) Z—CO—R³ wherein Z is any substituent capable of being involved in peptide bond formation preferably hydroxide, halide or azide, and R², R³, R⁵, X and n are as defined above. It will be appreciated that when R³ is a protected peptide, subsequent N-terminus extension may be carried out using peptide synthesis methods known in the art, such as deprotection and further peptide bond formation.

The process of the present invention can particularly be used for the production of cyclic compounds, for example cyclic peptides.

It will be appreciated that when R³ is C(R^(1′))(R⁹)—N(R¹⁰)(R¹¹), the compound of formula (II) can be reacted with one or more compounds of formula (V) in a stepwise direction.

The present invention therefore encompasses a compound of formula (VII);

wherein the variables are described above, preferably m is an integer of 1 to 50, preferably 1 to 30, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19 or 20; and R^(1′), R², R⁵, R⁹, R¹⁰, R¹¹, X and n are as defined above.

The compound of formula (VII) can be used in a process for the formation of a compound of formula (I) (illustrated below as (Ic));

when R¹⁴ is —[C(O)—C(R⁹)(R^(1′))—N(R¹⁰)—]_(m)—(R¹¹) and R^(1′), R², R⁶, R⁷, R⁹, R¹⁰, R¹¹, m, X and Y are as described above, comprising reaction of a compound of formula (VII) with a compound of formula (III) as described above. The compound of formula (I) can then be converted into a compound of formula (VI) by removal of the group R⁶ as described below. For the compound of formula (VII) and compounds of formula (I) or (VI) obtained therefrom, the substituents R¹ and R⁹ can be replaced by a group (═R¹) wherein R¹ is as described above.

It will be appreciated that when m is 3 or more, and R¹⁰ and R¹¹ are hydrogen, condensation can occur at the X—C(O)— functionality of the compound of formula (VII) to form a cyclised compound of formula (VIII);

wherein the variables are as defined above.

The present invention therefore provides a compound of formula (VIII). In addition, the invention provides a process for the production of a compound of formula (VIII) comprising cyclisation of a compound of formula (VII) wherein m is 3 or more. Reaction of the compound of formula (VIII) under reducing conditions (for example in the presence of lithium and liquid ammonia) results in the formation of a compound of formula (IX);

wherein R^(1′), R², R⁹, X and m are as defined above.

The present invention therefore provides a compound of formula (IX) and a process for the production of a compound of formula (IX) comprising the reduction of a compound of formula (VIII).

It will be appreciated that the process of the present invention can be carried out in solution. Alternatively, a compound of formula II may be attached to a resin via the group R⁵ and the peptide synthesis carried out via solid phase peptide synthesis. When R⁵ is a linker it can be a group OR¹³, N(R¹³)₂, CO₂R¹³ or Se, or an alkyl group having 1 to 4 carbons or a C₆₋₁₂ aryl group, said alkyl and aryl groups being optionally substituted with OR¹³, N(R¹³)₂, CO₂R¹³ or SR¹³. Alternatively, part of the synthesis may be carried out on the solid phase and part in solution.

The compound of formula II can be attached to and removed from a resin using methods known in the art.

Solid phase peptide synthesis using the process of the present invention may be carried out by using procedures attaching the carboxy-terminal to any resin known in the art. Examples of suitable resins include Wang, Merrifield, polyimide, 2-chlorotrityl, Rink, Knorr, DCHD, PAL and any other known in the art. Solid phase coupling partners such as BOP, PyBOP and DCC may be used, as well as any other suitable coupling partners known in the art.

A further feature of the first aspect is a process for the production of a compound of formula (VI)

from formula (I)

by the removal of R⁶ by any method known in the art, wherein X, Y, R², R³, R⁶ and R⁷ are as defined above. It will be appreciated that when R⁶ is hydrogen, the compound of formula (I) corresponds to the compound of formula (VI).

In particular, the removal of the group R⁶ may be carried out under reducing conditions such as under Birch conditions (i.e. with lithium and liquid ammonia). As it will be appreciated by the skilled person, the peptide produced by the process of the first aspect may be post modified by any suitable method known in the art. A second aspect of the present invention relates to the compounds described herein, including a compound of formula (II)

wherein X is O, S or NR⁸, where R⁸ is C₁₋₆ alkyl, C₆₋₁₂ aryl or hydrogen R² is independently selected from a C₁₋₁₀ branched or straight chain alkyl group, C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group, optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; R³ is as defined for R² or is hydrogen, or a group

or a group —C(R^(1′))(R⁹)—N(R¹⁰)(R¹¹) wherein R¹ is independently selected from a C₁₋₁₀ branched or straight chain alkyl group, C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₃R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂ wherein when Y is NR⁸, R⁸ and R^(1′) can together form a 4 to 7 membered ring, optionally substituted with CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more heteroatoms selected from O, S or N; R¹² is hydrogen, C₁₋₆ alkyl, C₆₋₁₂ aryl or N(R¹³)₂, wherein each occurrence of R¹³ is independently hydrogen, C₁₋₆ alkyl or C₆₋₁₂ aryl, R⁹ and R¹⁰ are independently hydrogen or a group as defined for R¹; or R⁹ and R¹⁰ can together form a 4 to 7 membered ring, optionally substituted with CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more heteroatoms selected from O, S or NR¹¹ is hydrogen or an amino protecting group preferably selected from a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a 2-nitrophenylsulphenyl group; R¹ is independently selected from C₁₋₁₀ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CO N(R¹³)₂, phenyl, imidazoyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂ and R^(4′) is a carboxyl protecting group or hydrogen and n is 0, 1, 2 or 3, m is 1-100; and R⁵ is a linker for attachment of formula (II) to a resin, a linked resin, or C₆₋₁₂ aryl, C₅₋₁₂ heteroalkyl or C₁₋₈ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; wherein when X═O, and R⁵ is phenyl, n is not 0 or 1.

Preferably R¹ and R² are independently selected from C₁₋₄ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CON(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂. More preferably R¹ and R² are independently selected from C₁ alkyl optionally substituted with OR¹³, SR¹³, CO₂R¹³, CO N(R¹³)₂, phenyl, imidazolyl, indolyl or hydroxyphenyl; C₂ alkyl optionally substituted with OR¹³, CO₂R¹³, CON(R¹³)₂ or SCH₃; C₃ alkyl NR¹³C(═NR¹³)N(R¹³)₂ or C₄ alkyl optionally substituted with N(R¹³)₂.

As set out above, R⁴ is a carboxyl protecting group, such as an ester group. In particular R⁴ is preferably methyl, ethyl, benzyl, t-butyl or phenyl. When R⁵ is a linker it can be OR¹³, N(R¹³)₂. CO₂R¹³ or SR¹³ or an alkyl group having 1 to 4 carbons or a C₆₋₁₂ aryl group, wherein the alkyl group and/or aryl group can be substituted with one or more of OR¹³, N(R¹³)₂, CO₂R¹³ or SR¹³.

The integer, m is preferably 1-50, more preferably 1 to 30, most preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The integer n is preferably 0 or 1.

When X is NR⁸, R⁸ is preferably C₁₋₄ alkyl, more preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or tert-butyl, phenyl, naphthyl, anthracenyl or phenanthracenyl, more preferably phenyl or hydrogen.

R¹³ is preferably hydrogen or C₁₋₄ alkyl, more preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or tert-butyl.

For the purposes of this invention, alkyl relates to both straight chain and branched, saturated or unsaturated alkyl radicals having, for example, 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms and most preferably 1 to 4 carbon atoms including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl n-pentyl, n-hexyl, n-heptyl, n-octyl. Alkyl therefore relates to a group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more carbon atoms. The term alkyl also encompasses cycloalkyl radicals of 3 to 12 carbon atoms, preferably 4 to 8 carbon atoms, and most preferably 5 to 6 carbon atoms including but not limited to cyclopropyl, cyclobutyl, CH₂-cyclopropyl, CH₂-cyclobutyl, cyclopentyl or cyclohexyl. Cycloalkyl groups may be optionally substituted or fused to one or more carbocyclyl or heterocyclyl group. Haloalkyl relates to an alkyl radical preferably having 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms substituted with one or more halide atoms for example CH₂CH₂Br, CF₃ or CCl₃. An alkyl group may be optionally interrupted by one or more O, S or NH groups, preferably one or more O atoms to form an alkoxy group. An alkyl group may be optionally interrupted by one or more double or triple bonds to form a group including but not limited to ethylene, n-propyl-1-ene, n-propyl-2-ene, isopropylene, ethynyl, 2-methylethynyl etc.

“Aryl” means an aromatic 6 to 12 membered hydrocarbon or heteroaryl containing one ring or being fused to one or more saturated or unsaturated rings including but not limited to phenyl, naphthyl, anthracenyl or phenanthracenyl. “Heteroaryl” means an aromatic 5 to 12 membered aryl containing one or more heteroatoms selected from N, O or S and containing one ring or being fused to one or more saturated or unsaturated rings including but not limited to furan, imidazole, indole, oxazole, purine, pyran, pyridine, pyrimidine, pyrrole, tetrahydrofuran, thiophene and triazole. The aryl and heteroaryl groups can be fully saturated, partially saturated or unsaturated.

Halogen means F, CI, Br or I, preferably F.

A third aspect of the invention relates to the use of a compound of formula (II) as defined in the first and/or second aspects of the invention in asymmetric synthesis.

All preferred features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

The present invention will now be illustrated by reference to one or more of the following non-limiting examples:

EXAMPLES

Example of a method for the production of a cyclic peptide.

Example of a method for N-terminal extension of a compound of formula (II).

Example of a method for use of the compound of formula (II) in solid phase synthesis

Example of a method to produce a thiomorpholinone template of formula (II)

Examples of Peptide Synthesis

(5R)-3-Methyl-5-ρhenyl-5,6-dihydro-2H-1,4-oxazin-2-one

(R)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) and ethyl pyruvate (2.67 mL, 24.1 mmol, 1.1 equiv.) were refluxed in trifluoroethanol (50 mL) over activated 4 molecular sieves (8.00 g) for 24 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo generated the crude product which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (7:3) to furnish the title compound as a white solid (1.70 g, 41%); mp 71.0-72.0° C. (lit 71.0-72.0° C.); v_((max)) (KBr) 3001 (C—H), 1734 (C═O), 1642 (C═N) cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.42-7.32 (5H, m, Ph), 4.89-4.80 (1H, m, PhCH), 4.56 (1H, dd, /4.49 Hz, T 11.55 Hz, 6β-H), 4.25 (1H, dd, J 10.97 Hz, T 11.51 Hz, 6α-H), 2.40 (3H, s, CH₃); δ_(C) (62.5 MHz, CDCl₃) 160.7, 155.9, 137.2, 129.4, 128.7, 127.5, 71.9, 60.1, 22.2; m/z (CL, NH₃), 189 (M⁺, 25%), 159 (12%), 130 (24%), 104 (100%), 90 (21%), and 78 (6%); HRMS for C₁₁H₁₁NO₂ requires 189.0787 found 189.0782. [α]_(D) ²⁰ −2560.0 (c 1.11 CHCl₃) (lit [α]_(D) ²⁰ −237.1 (c 1.11 CHCl₃)).

(3S,5R)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one

To a solution of (5R)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (1.70 g, 9.0 mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under an atmosphere of nitrogen was added PtO₂ (170 mg, 0.1 equiv.). The mixture was consecutively degassed and purged three times with hydrogen and then stirred for 5 hours under an atmosphere of hydrogen. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by recrystalisation in dichloromethane, diethyl ether and hexane to furnish the title compound as white needles (1.37 g, 80%); m.p. 82.0-83.0° C. (lit. m.p. 81.0-82.0° C.); v_((max))(KBr) 3314 (N—H), 2981 (C—H), 1739 (C═O), cm⁻¹, δ_(H) (250 MHz, CDCl₃) 7.44-7.33 (5H, m, Ph); 4.42-4.23 (3H, m, CHCH₂), 3.88 (1H, q, /6.76 Hz, CHCH₃), 1.86 (1H, br, NH), 1.50 (3H, d, J 6.16 Hz, CH₃); δ_(c) (62.5 MHz, CDCl₃) 170.7, 138.0, 129.3, 127.5, 127.2, 75.4, 58.2, 55.4, 19.0 m/z (CL, NH₃), 191 (M⁺, 7%), 147 (20%), 131 (65%), 104 (100%), 91 (20%), and 77 (12%); HRMS for C₁₁H₁₃NO₂ requires 191.0943 found 191.0940. [α]_(D) ²⁰ −92.9 (c 1.02 CHCl₃) (lit. for the enantiomer [α]_(D) ²⁰ +92.3 (c 0.84 CHCl₃)).

(3S,5R)-4-N-Acetyl-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

To a vigorously stirred mixture of (3S,5R)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (300 mg, 1.56 mmol), Na₂CO₃ (500 mg, 4.68 mmol, 3.0 equiv.) in anhydrous dichloromethane (30 mL) was added acetyl chloride (0.17 mL, 2.34 mmol, 1.5 equiv.) dropwise over 1 min. The resulting solution was stirred for 15 minutes under an atmosphere of nitrogen. The reaction was quenched by the addition of saturated Na₂CO₃ (20 mL), the aqueous phase was extracted with diethyl diethyl ether (3×10 mL) and the combined extracts were dried over MgSO₄. The solvents were removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with diethyl ether and dichloromethane (9:1) to furnish the title compound as fine colourless needles (270 mg, 74%); m.p. 82-85° C.; V_((max)) (KBr) 2943 (C—H), 1733 (C═O, lactone), 1647 (C═O, amide) cm^(″1); δ_(H) (250 MHz, DMSO-d) 7.42-7.36 (5H, m, Ph), 5.49 (0.4H, PhCH×0.4) 5.33 (0.6H, PhCH×0.6), 5.08-5.05 (1H, m, CH₃CH), 4.65 (2H, d, J=6.10 Hz, CH₂); 2.13 (1H, s, CH₃CON×1), 1.86 (2H, s, CH₃CON×2), 1.39 (3H, d, J=12.40 Hz CH₃CH); δ_(c) (62.5 MHz, DMSO-O 170.5, 169.8, 137.8, 129.4, 128.4, 127.1, 68.6, 55.5, 50.7, 49.7, 22.7, 19.0; ^(m)I_(τ) (CL, NH₃), 234 (MH⁺, 8%), 233 (M, 13%), 220 (4%), and 219 (100%); HRMS for C₁₃H₁₆NO₃ requires 234.1126, found 234.1130; [α]_(D) ²⁰−29.8 (c 1.16 CHCl₃).

N-Fmoc-L-alanine Acid Chloride

To a solution of N-Fmoc-L-alanine (2.00 g, 6.4 mmol, 1.0 equiv.) in anhydrous dichloromethane (40 mL) was added thionyl dichloride (4.70 mL, 64 mmol, 10 equiv.). The resulting mixture was refluxed for 2 hours under an atmosphere of nitrogen. The solvent and excess of thionyl chloride were removed in vacuo and the crude N-Fmoc-L-alanine acid chloride was partially purified by recrystallization from dichloromethane and hexane (1.74 g, 86%); m.p. 88-90° C. (lit. m.p. 112-114° C.); v_((max)) (KBr) 3328 (N—H). 3040 (C—H), 1778 (C═O, chloride), 1694 (C═O, carbamate), cm¹; δ_(H) (250 MHz. CDCl₃) 7.79-7.29 (8H, m. Fmoc). 5.22 (1H, d. J 8.0 Hz, NH), 4.67-4.38 (3H, m, CH₃CH×1, CHCH₂×2), 4.23 (1H, t, /6.52 Hz, CHCH₂); 1.55 (3H, d, J=7.28 Hz, CH₃CH); 8, (62.5 MHz, CDCl₃) 176.8, 158.2, 143.9, 141.7, 128.2, 127.5, 125.3, 120.5, 67.7, 59.1, 47.5, 17.7; ^(m)I_(z) (CL, NH₃), 330 (MH⁺, 48%), 258 (20%), 197 (65%), 154 (100%), 95 (20%), and 72 (12%); HRMS for C₁₈H₁₇ClNO₃ requires 330.1595 found 330.1592. [α]_(D) ²⁰+8.50 (c 1.30 CHCl₃) (lit. [α]_(D) ²⁴+4.03 (c 1.00 CH₂Cl₂)).

(3S,5R)—N—[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

To a vigorously stirred mixture of (3S,5R)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (600 mg, 3.14 mmol), Na₂CO₃ (1.70 g. 15.70 mmol, 5.0 equiv.) in 1:1 dichloromethane and water (40 mL) was added N-Fmoc-L-alanine acid chloride (1.26 g, 3.84 mmol, 1.2 equiv.) in dichloromethane (10 mL) dropwise over 5 min. The resulting solution was stirred for 2 hours. The aqueous phase was extracted with dichloromethane (3×15 mL). The combined extracts were washed with saturated Na₂CO₃ (50 mL), water (2×30 mL), brine (50 mL) and dried over MgSO₄. The solvents were removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:4) to furnish the title compound as fine colorless needles (1.21 g, 80%); m.p. 89-90° C.; V_((max)) (KBr) 3321 (N—H)₅ 2983 (C—H), 1741 (C═O, lactone), 1718 (C═O, carbamate), 1654 (C═O, amide) cm^(″1); δ_(H) (250 MHz, DMSO-d) 7.91-7.27 (13H, m, Fmoc×8, Ph×5), 5.50 (1H, br, NH), 5.07 (1H, m, PhCH), 4.90 (1H, m, NCHCH₃); 4.77 (1H, d, 77.0 Hz, Cc-PhCHCH₂), 4.57 (2H, m, CHCH₃NH×1, β-PhCHCH₂×1), 4.26 (1H, m, OCH₂CH), 4.19 (2H, m, OCH₂CH), 1.28 (3H, d, /4.O Hz₅ NCHCH₃), 1.10 (3H, d, /4.0 Hz, CHCH₃NH); δ_(c) (62.5 MHz, DMSO-d) 172.0, 170.0, 156.1, 144.2, 141.1, 136.2, 128.9, 128.3, 127.9, 127.4, 126.7, 125.7, 120.4, 66.0, 65.3, 53.0, 50.7, 47.8, 46.9, 18.6, 17.8; (Cl, NH₃), 508 (MNa⁺, 6%), 502 (MNH₄ ⁺, 45%), 487 (4%), and 485 (MH⁺, 100%); HRMS for C₂₉H₂₉N₂O₅ requires 485.2069. found 485.2060; [α]_(D) ²⁰−13.1 (c1.06 CHCl₃).

(5S)-3-Methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one

(S)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) and ethyl pyruvate (2.67 mL, 24.1 mmol, 1.1 equiv.) were refluxed in trifluoroethanol (50 mL) over activated 4 molecular sieves (8.00 g) for 24 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo generated the crude product which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (7:3) to furnish the title compound as a white solid (1.83 g, 44%); m.p. 70.0-71.0° C. (lit 71.0-72.0° C.); v_((max))(KBr) 3007 (C—H), 1735 (C═O), 1640 (C═N) cm⁻¹; δ_(H), (250 MHz, CDCl₃) 7.45-7.32 (5H, m, Ph), 4.88-4.81 (1H, m, PhCH), 4.56 (1H, dd, J 4.49 Hz, T 9.48 Hz, 6β-H), 4.25 (1H, dd/13.01 Hz, JT 14.99 Hz, 6α-H), 2.41 (3H, s, CH₃); δ_(c) (62.5 MHz CDCl₃) 160.7, 155.9, 137.2, 129.4, 128.7, 127.5, 71.9, 60.1, 22.2; m/z (CL, NH₃), 189 (M⁺, 25%), 159 (12%), 130 (24%), 104 (100%), 90 (21%), and 78 (6%); HRMS for C₁₁H_(n)NO₂ requires 189.0787 found 189.0782. [α]_(D) ²⁰ +253.0 (c 0.98 CHCl₃) (the enantiomer lit. [α]_(D) ²⁰ −237.1 (c 1.11 CHCl₃)).

(3R,5S)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one

To a solution of (5S)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (1.70 g, 9.0 mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under an atmosphere of nitrogen was added PtO₂ (170 mg, 0.1 equiv.). The mixture was consecutively degassed and purged three times with hydrogen and then stirred for 5 hours under an atmosphere of hydrogen. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by recrystallisation in dichloromethane, diethyl ether and hexane to furnish the title compound as white needles (1.26 g, 74%); m.p. 81.0-82.0° C. (lit. m.p. 81.0-82.0° C.); v_((max))(KBr) 3314 (N—H), 2981 (C—H), 1736 (C═O), cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.43-7.26 (5H, m, Ph); 4.42-4.23 (3H, m, CHCl₂), 3.88 (1H, q, /6.76 Hz, CHCH₃), 1.80 (1H, br, NH), 1.50 (3H, d, J 6.76 Hz, CH₃); δ_(c) (62.5 MHz, CDCl₃) 170.7, 138.1, 129.3, 129.1, 127.5, 75.4, 58.2, 55.4, 19.0; m/z (CL, NH₃), 192 (MH⁺, 30%), 147 (68%), 132 (64%), 104 (100%), and 91 (10%); HRMS for C₁₁H₁₃NO₂ requires 192.1025 found 192.1019. [α]_(D) ²⁰ +88.8 (c 0.96 CHCl₃) (lit. [α]_(D) ²⁰ +92.3 (c 0.84 CHCl₃)).

(3R,5S)—N—[N-Fmoc(S)alanyl]-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one

To a vigorously stirred mixture of (3R,5S)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (500 mg, 2.62 mmol), Na₂CO₃ (1.40 g, 13.3 mmol, 5.0 equiv.) in 1:1 dichloromethane and water (40 mL) was added N-Fmoc-L-alanine acid chloride (1.04 g, 3.14 mmol. 1.2 equiv.) in dichloromethane (10 mL) dropwise over 5 min. The resulting solution was stirred for 2 hours. The aqueous phase was extracted with dichloromethane (3×15 mL). The combined extracts were washed with saturated Na₂CO₃ (50 mL), water (2×30 mL brine (50 mL) and dried over MgSO₄. The solvents were removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:4) to furnish the title compound as fine colourless needles (1.02 g, 80%); m.p. 87-88° C.; v_((max)) (KBr) 3323 (N—H), 2982 (C—H), 1761 (C═O, lactone), 1717 (C═O, carbamate), 1656 (C═O, amide) cm^(″1); δ_(H) (400 MHz, DMSO-d, 110° C.) 7.84-7.29 (13H, m, Fmoc×8, Ph×5), 7.08 (1H, br, NH), 5.54 (1H, t, /5.88 Hz, PhCH), 4.96 (1H, q, /7.11 Hz, NCHCH₃); 4.68-4.60 (2H, m, PhCHCH₂), 4.41-4.32 (3H, m, CHCH₃NH×1, OCH₂CH×2), 4.22 (1H, t, /6.72 Hz OCH₂CH), 1.45 (3H, d, /7.15 Hz, NCHCH₃), 1.08 (3H, d, /6.71 Hz, CHCH₃NH); 6, (62.5 MHz, OMSO-d) 174.0, 172.8, 170.4, 169.6, 156.3, 144.1, 141.1, 137.7, 129.4, 128.7, 128.0, 127.4, 127.3, 127.1, 125.6, 120.5, 68.8, 66.0, 55.5, 55.3, 52.2, 51.4, 50.0, 47.2, 20.5, 18.6, 17.7, 17.1; ^(m)I_(z) (C.I. NH₃), 485 (MH⁺, 12%), 431 (8%), 381 (7%), 281 (15%) and 149 (100%); HRMS for C₂₉H₂₉N₂O₅ requires 485.2069. found 485.2076; [α]_(D) ²⁰+22.2 (c 0.94 CHCl₃).

(5R)-3-Isopropyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one

(R)-2-phenylglycinol (4) (2.00 g, 14.6 mmol, 1.0 equiv.) and ethyl 3-methyl-2-oxobutyrate (2.20 mL, 14.6 mmol. 1.0 equiv.) were refluxed in trifluoroethanol (30 mL) over activated 4 molecular sieves (7.0 g) for 24 hours. Filtration through a short pad of Celite® CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo generated the crude product which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (4:1) to furnish the title compound as a colourless oil (1.13 g, 36%); v_((max))(film) 2965 (C—H), 1740 (C═O), 1638 (C═N) cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.45-7.32 (5H, m, Ph), 4.90-4.83 (1H, m, PhCH), 4.55 (1H, dd, J 4.4 Hz, T 11.5 Hz, 6β-H), 4.13 (1H, dd/10.9 Hz, T 11.4 Hz, 6α-H), 3.32 (1H, m, CH(CH₃)₂), 1.25 (3H, d, J 5.0 Hz, CH(CH₃)₂×3), 1.22 (3H, d, /5.0 Hz, CH(CH₃)₂×3); δ_(c) (62.5 MHz, CDCl₃) 167.7, 155.7, 137.5, 129.3, 128.6, 127.4, 71.7, 59.7, 32.7, 20.7, 19.9: m/z (CL, NH₃), 220 (100%), 218 (MH⁺, 17%), 217 (27%), and 216 (25%); HRMS for C₁₃H₁₆NO₂ requires 218.1177 found 218.1181. [α]_(D) ²⁰-207.5 (c 1.17 CHCl₃).

(3S,5R)-3-Isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (39)

To a solution of (5R)-3-isopropyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (38) (1.03 g, 4.74 mmol, 1.0 equiv.) in anhydrous dichloromethane (50 mL) under an atmosphere of nitrogen was added PtO₂ (103 mg, 0.1 equiv.). The mixture was consecutively degassed and purged three times with hydrogen and then stirred for 5 hours under an atmosphere of hydrogen. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica eluting with petrol and diethyl ether (4:1) to furnish the title product as a waxy solid (662 mg, 64%); m.p. 61.5-62.5° C.; v_((max))(KBr) 3326 (N—H—), 2960 (C—H), 1733 (C═O), cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.47-7.35 (5H, m, Ph); 4.33-4.18 (3H, m, PhCHCH₂), 3.81 (1H, m, NHCH), 2.49, (1H, m, CH(CH₃)₂), 1.68 (1H, br, NH), 1.09 (3H, dd, J 6.75 Hz, CH(CH₃)₂×3), 1.05 (3H, dd, J 6.75, CH(CH₃)₂×3); δ_(c), (62.5 MHz, CDCl₃) 170.3, 138.5, 129.3, 129.1, 127.6, 74.9, 64.2, 57.3, 32.0, 19.4, 17.4; m/z (Cl, NH₃), 237 (MNH₄ ⁺, 5%), 220 (100%, MH⁺), 219 (22%), and 216 (6%); HRMS for C₁₃H₁₈NO₂ requires 220.1333 found 220.1338. [α]_(D) ²⁰ −92.1 (c 1.45 CHCl₃).

(3S,5R)—N—[N-Fmoc-(S)alanyl]-3-isopropyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

Method 1:

To a solution of (3S,5R)-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (150 mg, 0.68 mmol) in anhydrous dichloromethane (10 mL) was added N-Fmoc-L-alanine acid chloride (276 mg, 0.82 mmol, 1.2 equiv.) in anhydrous dichloromethane (5 mL). The resulting solution was stirred for 24 hours. The solvent was removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (2:3) to furnish the title product as colourless fine needles (95 ma, 27%).

Method 2:

To a vigorously stirred solution of (3S,5R)-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (200 mg, 0.91 mmol) and Na₂CO₃ (415 mg, 2.73 mmol, 3.0 equiv.) in anhydrous dichloromethane (15 mL) was added N-Fmoc-L-alanine acid chloride (428 mg, 1.36 mmol, 1.5 equiv.) in anhydrous dichloromethane (10 mL). The resulting mixture was stirred under nitrogen for 1 hour. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo furnished the crude material which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (3:7) to furnish the title product as colourless fine needles (380 mg, 82%); m.p. 72-75° C.; v_((max))(KBr) 3402 (N—H), 2969 (C—H), 1760 (C═O, lactone), 1718 (C═O, carbamate), 1654 (C═O, amide) cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.65-7.15 (13H, m, Fmoc×8, Ph×5), 5.46 (1H, br, NH), 5.03-4.98 (1H, m, PhCH), 4.75 (1H, d, /10.0 Hz CHCH(CH₃)₂), 4.32-4.13 (3H, m, PhCHCH₂ and OCH₂CH), 4.07-4.02 (1H, m, CHCH₃), 3.89-3.79 (2H, m, OCH₂CH), 2.18-2.00 (1H, m, CH(CH₃)₂), 1.21 (3H, d, J 5.5 Hz, CH(CH₃)₂×3), 1.19 (3-H, d, /5.0 Hz, CHCH₃), 1.02 (3H, d, J 6.5 Hz, CH(CH₃)₂×3); δ_(c) (62.5 MHz, CDCl₃) 174.1, 167.1, 153.7, 142.8, 140.2, 134.9, 128.8, 128.2, 127.2, 124.9, 124.1, 118.9, 66.3, 65.7, 55.2, 52.5, 47.5, 45.0, 31.6, 20.1, 18.2, 17.8; m/z (C₁, NH₃), 535 (MNa⁺, 73%), 513 (MH⁺, 100%), 334 (11%), 333 (53%), 328 (23%), and 311 (19%); (Cl.) HRMS for C₃₁H₃₃N₂O₅ requires 513.2381, found 513.2378. [α]_(D) ²⁰ −22.1 (c 1.05 CHCl₃).

N-Fmoc-L-Valine Acid Chloride

To a solution of N-Fmoc-L-alanine (3.00 g, 8.7 mmol, 1.0 equiv.) in anhydrous dichloromethane (40 mL) was added thionyl chloride (6.5 mL, 87 mmol, 10.0 equiv.). The resulting mixture was refluxed for 2 hours under an atmosphere of nitrogen. The solvent and excess of thionyl dichloride were removed in vacuo and the crude N-Fmoc-L-alanine acid chloride was partially purified by recrystallization from dichloromethane and hexane (2.50 g, 80%); m.p. 75-79° C. (lit. m.p. 111-112° C.); v_((max)) (K Br) 3317 (N—H), 2969 (C—H), 1788 (C═O, acid chloride), 1696 (C═O, carbamate), cm^(″1); δ_(H) (250 MHz, CDCl₃) 7.78-7.29 (8H, m, Fmoc), 5.20 (1H, d, J=9.5 Hz, NH), 4.55-4.33 (3H, m, CH₃CH×1, CHCH₂×2), 4.23 (1H, t, J=6.5 Hz, CHCH₂); 2.40 (1H, m, CH(CH₃)₂), 1.05 (3H, d, J 7.0 Hz, CH(CHg)₂×3), 0.95 (3H, d, /7.0 Hz, CH(CHb)₂×3); δ_(c) (62.5 MHz, CDCl₃) 175.4, 157.1, 143.9, 141.8, 128.2, 127.5, 125.3, 120.5, 68.2, 67.7, 47.5, 30.3 19.7, 17.4; [α]_(D) ²⁰+13.2 (c 1.05 CHCl₃) (lit. [α]_(D) ²⁴+5.5 (c 1.00 CH₂Cl₂)).

(3S,5R)—N—[N-Fmoc-(S)valinyl]-3-isopropyl-5-ρhenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

Method 1:

To a solution of (3S,5R)-3-wøpropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (200 mg, 0.91 mmol) in anhydrous dichloromethane (20 mL) was added N-Fmoc-L-valine acid chloride (391 mg. 1.10 mmol. 1.2 equiv.) in anhydrous dichloromethane (5 mL). The resulting solution was stirred for 24 hours. The solvent was removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (2:3) to furnish the title product as colourless fine needles (80 mg, 16%).

Method 2:

To a vigorously stirred solution of (3S,5R)-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (200 mg, 0.91 mmol) and Na₂CO₃ (415 mg, 2.73 mmol, 3.0 equiv.) in anhydrous dichloromethane (15 mL) was added N-Fmoc-L-valine acid chloride (533 mg, 1.36 mmol, 1.5 equiv.) in anhydrous dichloromethane (10 mL). The resulting mixture was stirred under nitrogen for 6 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo furnished the crude material which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:1) to furnish the title product as colourless fine needles (260 mg, 52%); m.p. 75-78° C.; V_((max))(KBr) 3421 (N—H), 2966 (C—H), 1763 (C═O, lactone), 1718 (C═O, carbamate), 1654 (C═O, amide) cm⁻¹; δ_(H) (400 MHz, DMSO-d₆, 120° C.) 7.83-7.25 (13H, m, Fmoc×8, Ph×5), 6.81 (1H, br, NH), 5.34 (1H, dd, /6.2 Hz, /10.8 Hz, PhCH), 4.77 (1H, d, /9.5 Hz, NCH), 4.58 (1H, dd, J 6.2 Hz, /12.5 Hz, PhCHCH₂×1), 4.47 (1H, dd, J 10.8 Hz, J 12.4 Hz, PhCHCH₂×1), 4.32-4.28 (1H, m, CH₃CH), 4.24-4.15 (3H, m, OCH₂Cl), 2.22-2.05 (2H, m, CH—(CH₃)₂×2), 1.18 (3H, d, J 6.5 Hz, CH(CH₃)₂×3), 0.96 (3H, d, J 6.5 Hz, CHCH₃), 0.84 (6H, t, J 7.0 Hz, CH(CH₃)₂×6); δ_(c) (62.5 MHz, CDCl₃) 174.5, 168.5, 155.5, 144.2, 141.7, 130.2, 129.6, 128.1, 127.4, 126.7, 126.4, 125.5, 120.4, 68.0, 67.1, 61.3, 57.7, 57.0, 47.4, 33.2, 32.6, 21.8, 20.1, 19.7, 18.4, 17.5; m/z (CL), 614 (100%), 576 (50%), 564 (9%), 541 (MH⁺), 519 (22%), and 503 (16%); HRMS for C₃₃H₃₇N₂O₅ requires 541.2692 found 541.2709. [α]_(D) ²⁰ −28.5 (c 0.56 CHCl₃).

Phenacyl N-tert-butoxycarboxyl-(S)-phenylalanate

To a solution of potassium hydroxide (0.64 g, 11.31 mmol) in methanol (15 mL) was added Boc-L-phenylalanine (3.00 g, 11.31 mmol). The resulting solution was stirred at room temperature for 3 hours. The solvent was removed and the crude material was dried in vacuo to yield white powder which was subsequently added in anhydrous N,N-dimethylformamide (15 mL) and treated with 2-bromoacetophenone (2.07 g, 13.57 mmol, 1.2 equiv.). The resulting solution was stirred at room temperature under nitrogen for 24 hours and was quenched by addition of water (20 mL). The precipitate can be either used as crude in next step or purified by recrystallization from diethyl ether yield the title compound as white fine needles (3.55 g, 82%); mp 140.0-141.0° C.; v_((max)) (KBr) 3395 (N—H), 2973 (C—H), 1757 (C═O, ester), 1715 (C═O, ketone), 1692 (C═O, carbamate) cm¹; δ_(H) (250 MHz, CDCl₃) 7.93-7.24 (IOH, m, Ph), 5.50 (1H, d, /16.4 Hz, OCH₂×1), 5.31 (1H, d, /16.4 Hz, OCH₂×1), 4.97 (1H, d, 8.1 Hz, NH), 4.75 (1H, dd, /7.2 Hz, r 6.1 Hz, CH), 3.36 (1H, dd, /5.4 Hz, r 14.1 Hz, PhCH, X 1), 3.14 (1H, dd, /7.1 Hz, /^(v) 14.0 Hz, PhCH₂×1), 1.40 (9H, s, f-butyl); δ_(c) (62.5 MHz, CDCl₃) 191.9 172.0, 155.6, 136.5, 134.4, 129.9, 129.3, 129.0, 128.2, 127.4, 80.4, 66.8, 54.7, 38.6, 28.7. [α]_(D) ²⁰−7.9 (c 1.01 CHCl₃).

(3S)-3-Benzyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2-one

To a suspension of phenacyl N-tert-butoxycarboxyl-(S)-phenylalanate (3.51 g. 9.16 mmol) in diethyl ether (150 mL) was added hydrogen bromide in acetic acid (33% w/w, 4.8 mL, 27.5 mmol, 3.0 equiv.). The resulting mixture was stirred under nitrogen for 3 hours during which time another portion of diethyl ether (100 mL) was added. The solid was separated by filtration through a sinter, washed with diethyl ether (2×30 mL) and dried in vacuo to furnish the amino ester hydrobromide which was subsequently dissolved in pH 5 acetate buffer (100 mL, 0.2 M, prepared from 70 parts 0.2 M aqueous sodium acetate and 30 parts 0.2 M aqueous acetic acid). The resulting mixture was stirred under nitrogen for 12 hours during which time yellow oil was formed. The precipitate can be either used as crude in next step or purified by flash column chromatography on silica, eluting with petrol and diethyl ether (3:2) to furnish the title compound as white solid (1.56 g, 65%); v_((max)) (KBr) 2932 (C—H), 1751 (C═O), cm^(″1); m.p. 57.0-59.0° C. (lit. 58.0-60.0° C.)⁶³; δ_(H) (250 MHz, CDCl₃) 7.90-7.18 (10H, m, Ph), 5.01 (1H, d, /14.2 Hz, CHCH₂O×1), 4.83 (H, m, CHCH₂Ph), 4.06 (1H, m, CHCH₂), 4.11 (1H, d, J=14.2 Hz, CHCH₂O×1), 3.46 (1H, dd, /5.45 Hz, /13.5 Hz, CHCH₂Ph×1), 3.32 (1H, dd, /5.45 Hz, /13.5 Hz, CHCH₂Ph×1); δ_(c) (62.5 MHz, CDCl₃) 169.0, 162.8, 136.6, 134.7, 131.7, 130.6, 129.2, 128.8, 127.6, 126.3, 67.8, 61.0, 39.4; % (CL, NH₃), 266 (32%), 265 (M⁺, 100%), 264 (42%), 263 (5%) and 262 (3%); HRMS for C₁₇H₁₅NO₂ requires 265.1103. found 265.1108. [α]_(D) ²⁰+85.1 (c 1.04 CHCl₃) (lit. [α]_(D) ²⁰+85.7 (c 2.00 CHCl₃)).

(3S,5R)-3-benzyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one

To a solution of (3S)-3-Benzyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2-one (1.56 g, 5.88 mmol, 1.0 equiv.) in anhydrous methanol (40 mL) under an atmosphere of nitrogen was added palladium on activated carbon (156 mg, 0.1 equiv. by mass). The mixture was consecutively degassed and purged three times with hydrogen and then stirred for 5 hours under an atmosphere of hydrogen. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica eluting with petrol and diethyl ether (4:1) to furnish the title product as a white solid (514 mg, 33%); m.p. 75.0-76.0° C. (lit. 76.0-78.0° C.); v_((max))(KBr) 3321 (N—H), 2949 (C—H), 1731 (C═O), cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.36-7.21 (10H, m, Ph), 4.34-4.15 (3H, m, PhCHCH₂), 3.97 (1H, dd, J 3.2, T 10.0 Hz, NHCH), 3.56, (1H, dd, J 3.2, J 13.6 Hz, CHPh×1), 3.00, (1H, dd, /10.0, T 13.6 Hz, CH₂Ph×1), 1.84 (1H, br, NH); δ_(H), (62.5 MHz, CDCl₃) 169.6, 138.0, 137.8, 129.8, 129.3, 129.2, 129.1, 127.5, 127.4, 75.3, 60.6, 57.9, 39.4; m/z (CL, NH₃), 268 (MH⁺, 40%), 267 (80%, M⁺), 223 (100%), and 209 (57%); HRMS for C₁₇H₁₇NO₂ requires 267.1259 found 267.1263. [α]_(D) ²⁰ −156.4 (c 1.02 CHCl₃) (lit. [α]_(D) ²⁰-157.9 (c 2.00 CHCl₃)).

(3S,5R)—N—[N-Fmoc-(S)-alanyl]-3-benzyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

To a vigorously stirred solution of (3S,5R)-3-benzyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (200 mg, 0.75 mmol) and Na₂CO₃ (239 mg, 2.25 mmol, 30 equiv.) in anhydrous dichloromethane (20 mL) was added N-Fmoc-L-alanine acid chloride (354 mg, 1.12 mmol, 1.5 equiv.) in anhydrous dichloromethane (6 mL). The resulting mixture was stirred under nitrogen for 4 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo furnished the crude product which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (2:3) to furnish the title product as colourless fine needles (150 mg, 36%); m.p. 87-89° C.; v_((max))(KBr) 3413 (N—H), 2981 (C—H), 1760 (C═O, lactone), 1718 (C═O, carbamate), 1656 (C═O, amide) cm⁻¹; δ_(H) (400 MHz, DMSO-d, 100° C.) 7.90-7.27 (18H, m, Fmoc×8, Ph×10), 7.07 (1H, br, NH), 5.47 (1H, dd, /4.8 Hz, /7.6 Hz, PhCH), 5.32 (1H, t, J 7.2 Hz, NCH), 4.82 (1H, dd, /7.6 Hz, J 12.4 Hz, PhCHCH₂×1), 4.71 (1H, dd, J 12 Hz, J′ 12.4 Hz, PhCHCH₂×1), 4.41 (1H, q, J 6.4 Hz, CH₃CH), 4.37-4.21 (3H, m, OCH₂CH), 3.15 (2H, d, 16.8 Hz, CH₂Ph), 1.21 (3H, d, /6.8 Hz, CH₃); δ_(c) (62.5 MHz, CDCl₃) 174.8, 170.8, 168.8, 159.6, 155.0, 142.7, 140.2, 128.9, 128.5, 127.9, 127.6, 127.4, 126.9, 126.6, 126.1, 124.2, 119.0, 66.2, 62.1, 61.0, 52.5, 47.8, 45.9, 33.8, 15.6; m/z (CL), 560 (M⁺), 502 (100%), 464 (43%), 426 (19%), and 414 (83%); HRMS for C₃₅H₃₂N₂O₅ requires 560.2303 found 560.2293. [α]_(D) ²⁰ −56.2 (c 1.01 CHCl₃).

(3S,5R)—N—[N-Fmoc-(S)-alanyl]-3-tert-butyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one

To a vigorously stirred solution of (3S,5R)-3-tert-butyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (150 mg, 0.63 mmol) in anhydrous dichloromethane (15 mL) and Na₂CO₃ (341 mg, 3.22 mmol, 5.0 equiv.) was added N-Fmoc-L-alanine acid chloride (298 mg, 0.95 mmol, 1.5 equiv.). The resulting mixture was stirred under an atmosphere of nitrogen for 18 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo gave the crude material which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:1) to furnish the title product as colourless fine needles (170 mg, 51%); m.p. 74-75° C.; v_((max))(KBr) 2976 (C—H), 1752 (C═O, lactone), 1724 (C═O, carbamate), 1662 (C═O, amide) cm⁻¹; δ_(H) (250 MHz, DMSO-d₆) 7.90-7.29 (13H, m, Fmoc×8, Ph×5), 5.39 (1H, br, PhCH), 4.62 (2H, br, CH₂O), 4.33 (1H, br, CH₃CH), 4.18 (3H, br, OCH₂CH), 1.23 (3H, d, /6.70 Hz, CHCH₃), 0.88 (9H, s, OCH); δ_(c), (62.5 MHz, DMSO-d₆) 175.9, 168.1, 155.8, 144.2, 144.1, 141.0, 137.1, 129.0, 128.0, 127.4, 125.8, 125.7, 120.4, 66.0, 62.2, 55.3, 53.0, 48.7, 46.9, 37.0, 28.6, 17.4; m/z (CL): 528 (15%), 527 (MB⁺, 34%), 526 (M, 100%), 470 (62%), and 414 (74%); HRMS for C₃₂H₃₄N₂O₅ requires 526.459 found 526.2457. [α]_(D) ²⁰ −10.9 (c 1.20 CHCl₃).

(3S,5R)—N—[N-Fmoc-(S)valinyl]-3-tert-butyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1, 4-oxazin-2-one

To a vigorously stirred solution of (3S,5R)-3-tert-butyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (75 mg, 0.32 mmol) in anhydrous dichloromethane (7 mL) and Na₂CO₃ (170 mg, 1.61 mmol, 5.0 equiv.) was added N-Fmoc-L-valine acid chloride (170 mg, 0.48 mmol, 1.5 equiv.). The resulting mixture was stirred under an atmosphere of nitrogen for 48 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo gave the crude material which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:1) to furnish the title product as colourless fine needles (32 mg, 18%); m.p. 71-73° C.; v_((max))(KBr) 2964 (C—H), 1752 (C═O, lactone), 1734 (C═O, carbamate), 1655 (C═O, amide) cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.77-7.25 (13H, m, Fmoc×8, Ph×5), 5.30 (1H, br, (CH₃)₂CHCH), 5.13 (1H, t, J 10.0 Hz, PhCH), 4.52-4.38 (2H, m, PhCHCH₂O), 4.29-4.21 (1H, n, OCH₂CH), 4.15 (3H, br, OCH₂CH×2 and CHC(CH₃)₃), 1.99-1.96 (1H, m, (CH₃)₂CH), 1.22 (9H, s, C(CH₃)₃); 0.97-0.84 (6H, m, (CH₃)₂CH); δ_(c) (62.5 MHz, CDCl₃) 175.9, 168.4, 155.4, 144.2, 141.7, 136.5, 130.2, 129.2, 128.1, 127.6, 126.4, 125.5, 120.4, 68.3, 67.1, 64.3, 57.6, 56.5, 47.5, 38.0, 32.7, 30.7, 20.2, 17.4; m/z (CL), 554 (MH⁺, 14%), 502 (100%), 464 (40%), 426 (20%), and 414 (74%); HRMS for C₃₄H₃₈N₂O₅ requires 554.2781 found 554.2790. [α]_(D) ²⁰ −17.5 (c 1.48 CHCl₃).

N-t-Boc-(S)-alanyl-N-(1-phenyl-2-hydroxylethyl)glycyl-(S)-alanine tert-butyl ester

To a suspension of L-alanine i-butyl ester hydrochloride (408 mg, 2.3 mmol) and (5S)-4-[N-?-Boc-(S)-alanyl]-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (0.78 g, 2.3 mmol) in anhydrous dioxane (20 mL) was added distilled triethylamine (0.32 mL, 2.3 mmol). The resulting mixture was stirred at room temperature vigorously for 5 days. Water (20 mL) was added and the mixture was extracted with diethyl ether (3×25 mL). The combined organic extracts were washed with brine (50 mL) and dried over MgSO₄. The solvents were removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol, diethyl ether, and methanol (10:10:1) to furnish the title compound as fine colourless needles (0.67 g, 59%); mp 69.0-70.0° C.; v_((max)) (KBr) 3312 (O—H), 2980 (C—H), 1738 (C═O, lactone), 1701 (C═O, carbamate), 1654 (C═O, amide) cm^(″1); δ_(H) (250 MHz, DMSO-O 8.69 (1H, d, J 7.0 Hz, NH), 7.39 (1H, d, J 7.0 Hz, NH), 7.40-7.26 (5H, m, Ph), 5.80-5.74 (1H, m, PhCH), 5.07-5.02 (1H, m, OH), 4.69 (1H, d, J 14.0 Hz, NCH₂×1), 4.13-4.04 (2H, m, CH₃CH×2), 3.96-3.91 (1H, m, HOCH₂), 3.77-3.74 (1H, m, HOCH₂), 3.63 (1H, d, J 18.0 Hz, NCH₂×1), 1.43 (18H, s, f-butyl×2), 1.23 (3H, d, J 13.0 Hz, CH₃), 1.12 (3H, d, J 13.0 Hz₅CH₃); δ_(c) (62.5 MHz, DMSO-J) 176.2, 172.0, 170.7, 156.1, 138.3, 129.0, 128.5, 127.5, 80.9, 78.6, 60.2, 57.5, 49.0, 46.7, 28.5, 27.9, 17.4, 16.9; % (CL, NH₃), 494 (MH⁺, 8%), 476 (6%), 376 (35%), 293 (21%), 249 (46%), and 44 (100%); HRMS for C₂₅H₄₀N₃O₇ requires 494.2856. found 494.2872. [α]_(D) ²⁰+24.3 (c 1.09 CHCl₃).

L-Alanine Tert-Butyl Ester

To a mixture of sodium carbonate (875 mg, 8.25 mmol, 5.0 equiv.) in deioned water (10 mL) and diethyl ether (10 mL) was added L-alanine tert-butyl ester hydrochloride (300 mg, 1.65 mmol, 1.0 equiv.). The resulting solution was stirred under an atmosphere of nitrogen for 1 hour. The aqueous phase was extracted with diethyl ether (3×10 mL) and the combined extracts were dried over MgSO₄. The solvent was removed in vacuo to settle the title compounds as a colourless oil (200 mg, 84%); v_((max)) (film) 3377 (N—H), 2978 (C—H), 1729 (C═O) cm^(″1); δ_(H)(250 MHz, CDCl₃) 3.42 (1H, q, J 7.02 Hz CH). 1.63 (2H, br, NH₂), 1.46 (9H, s, (CH₃)₃), 1.29 (3H, d, J 7.02 Hz, CH₃CH); δ_(c) (62.5 MHz, CDCl₃) 177.1, 81.2, 51.0, 28.4, 21.2; [α]_(D) ²⁰+3.7 (c 0.97 CHCl₃), (lit. [α]_(D) ²⁴+2.3 (c 1.00 CHCl₃)).

N-Fmoc-(S)-alanyl-N-((1R)-ρhenyl-2-hydroxylethyl)-(S)-alanyl-(S)-alanine tert-butyl ester

Method 1:

(3S,5R)—N—[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (300 mg, 0.62 mmol) and L-alanine tert-butyl ester (135 mg, 0.93 mmol, 1.5 equiv.) were dissolved in anhydrous dichloromethane (14 mL) in a doubly sealed PTFE cylinder and subjected to ultra-high pressure of 19 kbar for 48 hours. After releasing the pressure, the solution was filtered through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica, eluting with diethyl ether and dichloromethane (4:1) to furnish the starting material (33) (40 mg, 133%) and the title product as fine colourless needles (122 mg, 31%).

Method 2:

To a solution of L-alanine tert-butyl ester (466 mg, 3.21 mmol, 3.0 equvi.) in anhydrous dichloromethane (30 mL) was added trimethyl aluminium (1.87 mL, 3.75 mmol, 2 M in hexane, 3.5 equiv.) under an atmosphere of nitrogen. After 15 minutes, (3S,5R)—N—[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (520 mg, 1.07 mmol) in anhydrous dichloromethane (10 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of water (10 mL) and the organic phase was then washed with saturated copper sulphate (20 mL). The aqueous phase was extracted with diethyl ether (3×20 mL) and the combined organic extracts were washed with brine (50 mL) and dried over MgSO₄. The solvents were removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with diethyl ether and dichloromethane (4:1) to furnish the title compound as fine colourless needles (497 mg, 74%); m.p. 69-71° C.; v_((max)) (KBr) 3409 (O—H), 2979 (C—H), 1733 (C═O, lactone), 1718 (C═O, carbamate), 1646 (C═O, amide) cm^(″1); δ_(H) (250 MHz, OMSO-d) 7.96-7.34 (13H, m, Fmoc×8 and Ph×5), 7.90 (1H, d, /7.41 Hz, NH), 6.10 (1H, s, OH), 5.41-5.33 (1H, m, PhCH), 4.86 (1H, q, /6.76 Hz, CHCH₃), 4.40-4.24 (3H, m, OCH₂CH), 4.16-4.07 (2H, m, CH₂OH), 3.79 (1H, q, /6.95 Hz, CHCH₃), 3.63 (1171, q, /6.65 Hz, CHCH₃), 1.44 (3H, d, /6.12 Hz, CHCH₃), 1.40 (3H, d, /7.19 Hz, CHCH₃), 1.27 (9H, s, C(CHg)₃), 0.90 (3H, d, /7.42 Hz, CHCH₃); δ_(c) (62.5 MHz, OMSO-d) 173.5, 171.3, 170.3, 156.6, 144.1, 141.1, 137.6, 129.1, 128.7, 128.0, 127.6, 125.6, 121.7, 120.5, 80.2, 66.2, 60.9, 60.2, 48.7, 47.4, 46.9, 27.8, 17.4, 17.1, 14.9; % (CL, NH₃), 775 (100%), 630 (MH⁺, 25%), 485 (34%), and 408 (17%); HRMS for C₃₆H₄₄N₃O₇ requires 630.3170. found 630.3166; [α]_(D) ²⁰−41.2 (c 1.08 CHCl₃).

N-Acetyl-N-(1-phenyl-2-hydroxylethyl)alanyl-(S)-alanine tert-butyl ester

(3S,5R)-4-N-acetyl-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (68) (220 mg, 0.94 mmol) and L-alanine tert-butyl ester (66) (205 mg, 1.42 mmol, 1.5 equiv.) were dissolved in anhydrous dichloromethane (14 mL) in a doubly sealed PTFE cylinder and subjected to ultra-high pressure of 19 kbar for 48 hours. After releasing the pressure, the solution was filtered through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica eluting with diethyl ether and dichloromethane (4:1) to furnish the title product as fine colourless needles (120 mg, 34%); m.p. 38-40° C.; v_((max))(KBr) 3411 (O—H), 2980 (C—H), 1735 (C═O, lactone), 1646 (C═O, amide) cm⁻¹; δ_(H) (250 MHz, DMSO-d) 8.06 (0.5H, d, /6.95 Hz, NH×0.5), 7.55-7.24 (5H, m, Ph), 6.54 (0.5H, d, J 6.38 Hz, NH×0.5), 5.82 (0.5H, t, /4.88 Hz, OH×0.5), 5.14-5.06 (1H, m, PhCH), 4.83 (0.5H, t, J 6.56 Hz, OH×0.5), 4.35 (0.5H, q, J 6.99 Hz, CH₃CH×0.5), 4.08-4.03 (2H, m, CH₂OH), 3.98-3.76 (1.5H, m, CHCH₃×0.5 and CHCH₃×1), 2.23 (1.5H, s, COCH₃), 2.14 (1.5H, s, COCH₃), 1.46-1.39 (12H, m, CHCH₃×3 and C(CH₃)₃×9), 1.19 (1.5H, d, /7.15 Hz, CHCH₃×1.5), 0.97 (1.5H, d, J 7.23 Hz, CHCH₃×1.5); δ_(c) (62.5 MHz, DMSO-d) 171.8, 171.7, 171.0, 170.8, 170.2, 140.0, 138.2, 129.1, 128.2, 128.0, 126.8, 80.7, 62.7, 62.5, 60.8, 56.3, 53.4, 48.8, 48.7, 27.9, 17.5, 16.9, 16.7, 15.2; m/z (CL, NH₃), 379 (MH⁺, 70%), 361 (18%), 305 (20%), 249 (38%), 234 (100%), and 219 (40%); HRMS for C₂₀H₃₁N₂O₅ requires 379.2225 found 379.2240; [α]_(D) ²⁰ −37.0 (c 1.06 CHCl₃).

N-Fmoc-(S)-alanyl-N-((1S)-phenyl-2-hydroxyl-ethyl)-(R)-alanyl-(S)-alanine tert-butyl ester

To a solution of L-alanine tert-butyl ester (251 mg, 1.73 mmol, 3.0 equiv.) in anhydrous dichloromethane (25 mL) was added trimethyl aluminium (2.03 mL, 2.03 mmol, 2 M in hexane, 3.5 equiv.) under an atmosphere of nitrogen. After 15 minutes, (3R,5S)—N—[N-Fmoc-(S)alanyl]-3-methyl-5-ρhenyl˜3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (280 mg, 0.58 mmol) in anhydrous dichloromethane (8 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of water (7 mL) and the organic phase was then washed with saturated copper sulphate (15 mL). The aqueous phase was extracted with diethyl ether (3×20 mL) and the combined organic extracts were dried over MgSO₄. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:4) to furnish the title compound as fine colourless needles (226 mg, 62%); m.p. 87-88° C.; v_((max)) (KBr) 3410 (O—H), 2980 (C—H), 1727 (C═O). 1654 (C═O) cm^(″1); δ_(H) (250 MHz, DMSO-d), 8.35 (0.5H, d, /6.18 Hz, NH) 7.91-7.12 (13H, m, Fmoc×8 and Ph×5), 7.65 (0.51-1, d, /7.14 Hz, NH), 7.55 (0.5H, d, /7.32 Hz, NH). 6.39 (0.5H, d, /7.45 Hz, NH), 5.21-5.18 (0.5H, m, PhCH), 5.03-5.01 (0.5H, m, PhCH), 4.94-4.92 (0.5H, m, NHCHCH₃), 4.80-4.78 (0.5H, m, NHCHCH₃), 4.65-4.62 (0.5H, m, NCHCH₃), 4.27-4.18 (3.5H, m, OCH₂CH, and Cl₂O×0.5), 4.09-3.93 (2.51-1, in, CH₂OH×1.5 and NHCHCH₃), 3.62-3.60 (0.5H, in, NCHCH₃×1), 1.44-1.36 (13.5H, s, (CEb)₃×9, CHCH₃×4.5), 1.23 (1.5H, d, /6.63 Hz, CHCH₃), 1.13 (1.51-1, d, /7.35 Hz, CHCH₃), 0.92 (1.5H, d, /7.07 Hz, CHCH₃); δ_(c) (62.5 MHz, OMSO-d) 173.4, 172.9, 171.9, 171.6, 169.7, 156.0, 144.2, 144.1, 141.1, 137.2, 129.0, 128.3, 128.0, 127.7, 127.4, 127.3, 125.7, 120.5, 81.2, 80.8, 66.1, 61.6, 55.3, 53.6, 48.6, 48.3, 47.5, 47.0, 27.9, 18.5, 18.2, 17.7, 17.1, 15.4; ^(m)I_(z) (CL, NH₃), 775 (100%), 629 (M⁺, 36%), 457 (58%), 345 (27%), 231 (33%) and 178 (100%); HRMS for C₃₆H₄₃N₃O₇ requires 629.3092. found 629.3093; [α]_(D) ²⁰+14.20 (c 1.15 CHCl₃).

L-Valine Tert-Butyl Ester

To a mixture of sodium carbonate (1.20 g, 9.54 mmol, 5.0 equiv.) in deioned water (20 mL) and diethyl ether (20 mL) was added L-valine tert-butyl ester hydrochloride (400 mg, 1.71 mmol, 1.0 equiv.). The resulting solution was stirred for 2 hours under an atmosphere of nitrogen. The aqueous phase was extracted with diethyl ether (3×15 mL) and the combined extracts were dried over MgSO₄. The solvent was removed in vacuo to settle the title compound as colourless oil (320 mg, 97%); v_((max)) (film) 3393 (N—H), 2967 (C—H), 1728 (C═O) cm^(″1); δ_(H) (250 MHz, CDCl₃) 3.16 (1H, d, J 4.79 Hz, CHNH₂), 2.04-1.97 (1H, m, CH(CH₃)₂) 1.47 (9H, s, (CH₃)₃), 1.38 (2H, s, NH₂), 0.97 (3H, d, /6.88 Hz, (CH)CH×3), 0.90 (3H, d, J 6.86 Hz, (CH₃)₂CH×3); δ_(c) (62.5 MHz, CDCl₃) 175.3, 81.2, 60.7 32.6, 28.5, 19.7, 17.4; [α]_(D) ²⁰+26.4 (c CHCl₃), (lit. [α]_(D) ²⁴+25.3 (c 1.00 CHCl₃)).

N-Fmoc-(S)-a\any\-N-(1-phenyl-2-hydroxylethyl)-(5)-alanyl-(5)-valine tert-butyl ester (74)

To a solution of L-valine tert-huiyl ester (161 mg, 0.93 mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added trimethyl aluminium (0.55 mL, 1.09 mmol, 2 M in hexane, 3.5 equiv.) under an atmosphere of nitrogen. After 15 minutes, (3S,5R)—[N(N-Fmoc-(S)alanyl]-3-methyl-5-ρhenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (150 mg, 0.31 mmol) in anhydrous dichloromethane (5 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of saturated copper sulphate (20 mL) and the mixture was extracted with diethyl ether (3×20 mL). The combined organic extracts were dried over MgSO₄. The solvents were removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with diethyl ether and dichloromethane (9:1) to furnish the title compound as fine colourless needles (128 mg, 63%); m.p. 75.0-76.0° C.; v_((max)) (KBr) 3411 (O—H), 2973 (C—H), 1718 (C═O). 1654 (C═O) cm¹; δ_(H) (250 MHz, DMSO-O 7.96 d, /7.4 Hz, NH), 7.97-7.29 (13H, m, Fmoc×8 and Ph×5), 5.43 (1H, br, OH), 5.35 (1H, t, /4.2 Hz, PhCH), 4.91 (1H, t, /6.8 Hz, CHCH₃), 4.43-4.40 (1H, m, OCH₂CH), 4.31-4.21 (2H, m, OCH₂CH), 4.20-4.05 (2H, m, HOCH₂), 3.79-3.71 (1H, m, CHCH(CH₃)₂), 3.70-3.68 (1H, m, CHCH₃), 1.81-1.76 (1H, m, CH(CH₃)₂), 1.46-1.34 (6H, m, CHCH₃×2) 1.31 (9H, s, f-butyl), 0.68-0.64 (6H, m, CH(CHg)₂); δ_(c) (62.5 MHz, OMSO-d) 173.1, 170.1, 170.0, 156.4, 144.2, 141.1, 137.6, 129.1, 128.7, 128.3, 128.0, 127.4, 125.7, 124.5, 80.5, 66.4, 63.2, 61.2, 58.1, 53.4, 47.1, 30.1, 27.9, 18.8, 18.4, 17.8, 15.1; ^(m)I_(z) (CL, NH₃), 657 (M⁺, 34%), 579 (40%), 427 (59%), 363 (26%), and 244 (100%); FIRMS for C₃₈H₄₇N₃O₇ requires 657.3404. found 657.3398; [α]_(D) ²⁰−38.7 (c 0.96 CHCl₃).

t-Butyl N—[(S)-2-hydroxy-1-phenylethyl]-(5)-valinyl-(5)-alanate

To a solution of L-alanine tert-butyl ester (180 mg, 1.24 mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added trimethyl aluminium (0.56 mL, 1.12 mmol, 2 M in hexane, 2.7 equiv.) under an atmosphere of nitrogen. After 15 minutes. (3S,5R)-3-wopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (91 mg, 0.41 mmol) in anhydrous dichloromethane (5 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of saturated copper sulphate (15 mL) and the mixture extracted with diethyl ether (3×15 mL). The combined organic extracts were dried over MgSO₄. The solvents were removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (9:1 then 1:20) to furnish the starting material (60 mg, 60%) and the title compound as light yellow oil (50 mg, 33%); v_((max)) (film) 3328 (O—H), 2977 (C—H), 1733 (C═O, ester), 1651 (C═O, amide) cm^(″1); δ_(H) (250 MHz, CDCl₃) 7.73 (1H, d, J 8.6 Hz, NH), 7.30-7.18 (5H, m, Ph), 4.54-4.78 (1H, m, CHCH₃), 3.74-3.68 (1H, m, PhCH), 3.59-3.50 (2H, m, HOCH₂), 2.79 (1H, d, 74.8 Hz, CHCH(CH₃)₂), 1.97-1.93 (1H, m, CH(CH₃)₂), 1.41 (9H, s, t-butyl), 1.29 (3H, d, /8.7 Hz, CH₃), 0.81 (31-1, d, 14.4 Hz, CH(CH₃)₂), 0.78 (3H, d, /4.4 Hz, CH(CH₃)₂); 6, (62.5 MHz, CDCl₃) 174.0, 140.8, 128.8, 128.1, 127.7, 125.9, 82.7, 67.6, 66.9, 64.6, 48.4, 31.8, 28.4, 20.0, 19.2, 18.2; ^(m)I_(z) (CL, NH₃), 365 (MH⁺, 52%), 339 (29%), 333 (76%), 291 (28%), 277 (100%) and 263 (10%); HRMS for C₂₀H₃₃N₂O₄ requires 365.2432. found 365.2430. [α]_(D) ²⁰−80.2 (c 0.98 CHCl₃).

Potassium N-benzylidenyl-(S)-valinate

To a solution of potassium hydroxide (958 mg, 17.10 mmol) in methanol (15 mL) was added L-valine (2.00 g, 17.10 mmol). The resulting solution was stirred under nitrogen for 3 hours. The solvent was removed in vacuo before benzaldehyde (2.61 mL, 25.70 mmol 2.0 equiv.) in anhydrous pentane (30 mL) was added and the mixture was azeotropic distillated under nitrogen for 8 hours. The precipitate was collected and dried in high vacuo to furnish the title compound as a white solid (3.85 g, 93%), V_((max)) (KBr) 2956 (C—H), 1594 (C═O) cm^(″1); δ_(H)(250 MHz, OMSO-d), 8.19 (1H, s, CH═N), 7.75-7.71 (2H, m, Ph×2), 7.44-7.40 (3H, m, Ph×3), 3.26 (1H, d, J 7.24, CHCH(CH₃)₂), 2.21 (1H, m, CHCH(CH₃)₂), 0.86 (3H, d, J 6.72 Hz, CHCH(CH₃)₂×3), 0.79 (31-1, d, /6.72 Hz, CHCH(CH₃)₂×3); 6, (62.5 MHz, DMSO-d) 174.7, 159.0, 137.1, 130.3, 128.8, 128.1, 84.8, 31.4, 20.8, 19.5.

N—[N-Fmoc-(S)-alanyl)]-3(S′)-isopropyl-5(R,S)-phenyl oxazolidinone

To the stirring suspension of potassium N-benzylidenyl-5-valinate (122 mg, 0.50 mmol) in anhydrous dichloromethane (20 mL) was added Fmoc-L-alanine acid chloride (166 mg, 0.50 mmol) under nitrogen at 0° C. The resulting mixture was stirred for 4 hours at 0° C. and for another 12 hours at room temperature. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol:diethyl ether (1:1) to furnish the title compound as fine colourless needles (174 mg, 70%); m.p. 69.0-71.5° C.; v_((maX)) (KBr) 3400 (N—H), 2966 (C—H), 1802 (C═O, lactone), 1718 (C═O, carbamate), 1674 (C═O, amide) cm¹; δ_(H) (250 MHz, OMSO-d) 7.70-7.17 (13H, m, Fmoc×8 and Ph×5), 6.45-6.43 (0.3H, m, PhCH×0.3), 5.36-5.28 (0.7H, m, PhCH×0.7), 4.64-4.47 (1H, m, CHCH₃×0.7 and CHCH(CH₃)₂×0.3), 4.47-4.09 (3H, m, CHCH₂), 4.01-3.82 (1H, m, CHCH₃×0.3 and CHCH(CH₃)₂×0.7), 2.83-2.55 (0.3H, m, CH(C₁₋₁₃)₂×0.3), 2.20-2.05 (0.7H, m, CH(CH₃)₂×0.7), 1.43-1.40 (3H, m, CHCH₃), 1.26-1.13 (3H, m, CH(CHs)₂×3) 1.02-0.91 (3H, m, CH(CH₃)₂×3), 0.87-0.80 (3H, m, CHCH₃); δ_(c) (62.5 MHz, CDCl₃) 177.5, 171.3, 169.5, 166.1, 156.0, 144.1, 141.7, 136.3, 131.7, 130.6, 130.2, 129.1, 128.2, 127.5, 127.0, 125.5, 120.4, 91.5, 70.2, 67.5, 61.8, 49.3, 47.5, 46.2, 34.2, 31.1, 30.7, 19.3, 18.9, 18.3, 17.7, 16.7; % (Cl, NH₃), 498 (M⁺, 35%), 476 (47%), 463 (44%), 425 (23%), and 413 (100%); HRMS for C₃₀H₃₀N₂O₅ requires 498.2155. found 498.2151.

t-Butyl Fmoc-(S)-ala-(S)-val-(S)-alanate (79)

To a solution of L-alanine tert-butyl ester (114 mg, 0.78 mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added trimethyl aluminium (0.46 mL, 0.91 mmol, 2 M in hexane, 3.5 equiv.) under an atmosphere of nitrogen. After 15 minutes, N—[N-Fmoc-(S)-alanyl)]-3(S)-wopropyl-5(R,S)-phenyl oxazolidinone (130 mg, 0.26 mmol) in anhydrous dichloromethane (5 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of saturated copper sulphate (20 mL) and the mixture was extracted with diethyl ether (3×20 mL). The combined organic extracts were dried over MgSO₄. The solvents were removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol:diethyl ether (1:9) to furnish the title compound as fine colourless needles (76 mg. 54%); mp 153.0-155.0° C.; v_((max)) (KBr) 3292 (N—H), 2974 (C—H), 1734 (C═O, ester), 1696 (C═O, carbamate), 1645 (C═O, amide) cm^(″1); δ_(H) (250 MHz, DMSO-d) 8.37 (0.5H, d, /6.42 Hz, NH), 8.18 (0.5H, d, /6.81 Hz, NH), 7.95 (0.5H, d, J 7.56 Hz, NH×0.5), 7.63 (0.5H, d, /7.48 Hz, NH×0.5), 7.96-7.35 (8H, m, Fmoc), 4.31-4.13 (5H, m, CHCH₃×2 and CHCH₂), 2.03 (1H, q, /6.57 Hz, CH(CH₃)₂), 1.42 (9H, s, f-butyl), 1.28-1.25 (6H, m, 2×CH₃) 0.94-0.85 (6H, m, CH(CHb)₂); δ_(c) (62.5 MHz, DMSO-J) 173.1, 172.6, 171.9, 170.8, 156.1, 144.1 141.1, 128.0, 127.4, 125.6, 120.5, 80.7, 66.0, 57.4, 57.0, 50.4, 48.7, 47.0, 31.5, 31.0, 30.8, 27.9, 19.5, 18.7, 18.3, 17.9, 17.4, 17.2; ^(m)I_(z) (CL. NH₃), 537 (M⁺, 12%), 502 (100%), 464 (50%), 426 (21%), and 414 (86%); FIRMS for C₃₀H₃₉N₃O₆ requires 537.2829. found 537.2836. [α]_(D) ²⁰−6.2 (c 0.57 CHCl₃).

N—[N-Fmoc-(S)-valinyl)]-3 (S)-isopropyl-5 (R,S)-phenyl oxazolidinone

To the stirring suspension of potassium N-benzylidenyl-(5)-valinate (123 mg, 0.46 mmol) in anhydrous dichloromethane (20 mL) was added Fmoc-L-valine acid chloride (164 mg, 0.46 mmol) under nitrogen at 0° C. The resulting mixture was stirred for 4 hours at 0° C. and for another 12 hours at room temperature. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol:diethyl ether (3:2) to furnish the title compound as fine colourless needles (183 mg, 75%); mp 76.0-78.0° C.; v_((max)) (KBr) 3326 (N—H), 2966 (C—H), 1802 (C═O, lactone), 1722 (C═O, carbamate), 1663 (C═O, amide) cm^(″1); δ_(H) (250 MHz, DMSO-d) 7.81-7.11 (14H, m. NH×1, Fmoc×8 and Ph×5), 6.4 (0.5H, s, PhCH×0.5), 4.98 (0.5H, s. PhCH×0.5), 4.48-4.76 (5H, m, CHCH(CH₃)₂×2 and CHCH₂×3), 2.57-2.54 (1H, m, CH(CH₃)₂), 2.24-2.10 (1H, m, CH(CHs)₂), 1.28-0.63 (12H, m, CH(CH₃)₂×2); δ_(c) (62.5 MHz, DMSO-d) 170.3, 170, 169.7, 156.9, 144.1, 141.0, 137.5, 130.9, 129.9, 129.5, 128.5, 128.0, 127.7, 127.4, 125.8, 120.5, 90.6, 66.2 61.3, 60.9, 58.3, 46.9, 34.7, 32.6, 30.8, 30.3, 19.7, 18.7, 18.3, 17.2, 16.7, 16.4; % (CL, NH₃), 526 (M⁺, 10%), 331 (40%), 230 (7%) and 178 (100%); HRMS for C₃₂H₃₄N₂O₅ requires 526.2459. found 526.2466.

Tri-L-alanine

To a solution of N-Fmoc-(S)-alanyl-N-((1R)-phenyl-2-hydroxylethyl)-(S)-alanyl-(S)-alanine tert-butyl ester (235 mg, 0.37 mmol) and tert-butanol (0.12 mL, 1.20 mmol, 3.0 equiv.) in liquid ammonia (15 mL) and anhydrous tetrahydrofuran (10 mL) was added lithium (23 mg, 3.70 mmol, 10.0 equiv.) at −78° C. under an atmosphere of nitrogen. The resulting solution was stirred until the blue colour was disappeared and then warmed to room temperature to evaporate all the liquid ammonia. A mixture of water (15 mL) and diethyl ether (10 mL) was added and the aqueous phase was extracted with diethyl ether (3×10 mL). Water was removed in vacuo and the crude product purified first by acidic ion exchange chromatography and then by flash column chromatography on silica, eluting with methanol and water (7:3) to furnish the title compound as fine colourless needles (71 mg, 84%), v_((max)) (KBr) 3276 (N—H), 2985 (C—H), 1645 (C═O), 1592 (C═O), 1531 (C═O) cm^(″1); δ_(H) (250 MHz, D₂O), 4.53 (1H, q, /9.37, CH), 4.03 (2H, q, /7.22 Hz, CH×2), 1.23 (3H, d, J 7.14, CH₃), 1.09 (3H, d, J 7.21, CH₃), 1.02 (3H, d, /7.24 Hz, CH₃); δ_(c) (62.5 MHz, D₂O) 180.1, 173.9, 170.9, 51.3, 50.1, 49.2, 17.7, 16.9, 16.7; ^(m)I_(z) (CL, NH₃), 231 (M⁺, 40%), 230 (100%), and 228 (35%); HRMS for C₉H₁₇H₃O₄ requires 231.1215 found 231.1209; [α]_(D) ²⁰−72.8 (c 1.01 H₂O) (commercial one [α]_(D)2^(j)0^(n)−73.2 (c 1.02 H₂O)).

(5S)-3-Methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one(35)⁶¹

(S)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) (34) and ethyl pyruvate (2.67 mL, 24.1 mmol, 1 equiv.) were refluxed in trifluoroethanol (50 mL) over activated 4 molecular sieves (8.00 g) for 24 hours. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo delivered the crude product which was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (7:3) to furnish the title compound as a white solid (1.83 g, 44%); m.p. 70.0-71.0° C. (lit 71.0-72.0° C.); v_((max))(KBr) 3007 (C—H), 1735 (C═O), 1640 (C═N) cm⁻¹; δ_(H) (250 MHz, CDCl₃) 7.45-7.32 (5H, m. Ph), 4.88-4.81 (1H, m, PhCH), 4.56 (1H, dd, J 4.49 Hz, T 9.48 Hz, 6β-H), 4.25 (1H, dd J 13.01 Hz, T 14.99 Hz, 6α-H), 2.41 (3H, s, CH₃); δ_(c) (62.5 MHz, CDCl₃) 160.7, 155.9, 137.2, 129.4, 128.7, 127.5, 71.9, 60.1, 22.2; m/z (CL, NH₃), 189 (M⁺, 25%), 159 (12%), 130 (24%), 104 (100%), 90 (21%), and 78 (6%); HRMS for C₁₁H₁₁NO₂ requires 189.0787 found 189, 0782. [α]_(D) ²⁰ +253.0 (c 0.98 CHCl₃) (the enantiomer lit. [α]^(D20) −237.1 (c 1.11 CHCl₃)).

(3R,5S)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (36)

To a solution of (5S)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (35) (1.70 g, 9.0 mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under an atmosphere of nitrogen was added PtO₂ (170 mg, 0.1 equiv.). The mixture was consecutively degassed and purged three times with hydrogen and then stirred for 5 hours under an atmosphere of hydrogen. Filtration through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by recrystallization from dichloromethane, diethyl ether and hexane to furnish the title compound as a colourless needles (1.26 g, 74%); m.p. 81.0-82.0° C. (lit. m.p. 81.0-82.0° C.); v_((max))(KBr) 3314 (N—H), 2981 (C—H), 1736 (C═O), cm⁻¹, δ_(H) (250 MHz, CDCl₃) 7.43-7.26 (5H, m, Ph); 4.42-4.23 (3H, m, CHCH₂), 3.88 (1H, q, /6.76 Hz, CHCH₃), 1.80 (1H, br. NH), 1.50 (3H, d, J 6.76 Hz, CH₃); δ_(c) (62.5 MHz, CDCl₃) 170.7, 138.1, 129.3, 129.1, 127.5, 75.4, 58.2, 55.4, 19.0; m/z (CL, NH₃), 192 (MH⁺, 30%), 147 (68%), 132 (64%), 104 (100%), and 91 (10%); HRMS for C₁₁H₁₃NO₂ requires 192.1025 found 192.1019. [α]_(D) ²⁰ +88.8 (c 0.96 CHCl₃) (lit. [α]_(D) ²⁰ +92.3 (c 0.84 CHCl₃)).

(3R,5S)—N—[N-Fmoc-(S)-alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (37)

To a vigorously stirred mixture of (3R,5S)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (36) (500 mg. 2.62 mmol), Na₂CO₃ (1.40 g, 13.3 mmol, 5.0 equiv.) in 1:1 dichloromethane and water (40 mL) was added N-Fmoc-L-alanine acid chloride (1.04 g, 3.14 mmol, 1.2 equiv.) in dichloromethane (10 mL) dropwise over 5 min. The resulting solution was stirred for 2 hours. The aqueous phase was extracted with dichloromethane (3×15 mL). The combined extracts were washed with saturated Na₂CO₃ (50 mL), water (2×30 mL), brine (50 mL) and dried over MgSO₄. The solvents were removed in vacuo and the crude material was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:4) to furnish the title compound as fine colourless needles (1.02 g, 80%); m.p. 87-88° C.; v_((max)) (KBr) 3323 (N—H), 2982 (C—H), 1761 (C═O, lactone), 1717 (C═O, carbamate), 1656 (C═O, amide) cm^(″1); δ_(H) (400 MHz, DMSO-J, 110° C.) 7.84-7.29 (13H, m, Fmoc×8, Ph×5), 7.08 (1H, br, NH), 5.54 (1H, t, /5.88 Hz, PhCH), 4.96 (1H, q, /7.11 Hz, NCHCH₃); 4.68-4.60 (2H, m, PhCHCH₂), 4.41-4.32 (3H, m, CHCH₃NH×1, OCH₂CH×2), 4.22 (1H, t, /6.72 Hz OCH₂CH), 1.45 (3H, d, /7.15 Hz₅NCHCH₃), 1.08 (3H, d, /6.71 Hz, CHCH₃NH); δ_(c) (62.5 MHz, OMSO-d) 174.0, 172.8, 170.4, 169.6, 156.3, 144.1, 141.1, 137.7, 129.4, 128.7, 128.0, 127.4, 127.3, 127.1, 125.6, 120.5, 68.8, 66.0, 55.5, 55.3, 52.2, 51.4, 50.0, 47.2, 20.5, 18.6, 17.7, 17.1; ′7_(Z) (C.I., NH₃), 485 (MH⁺, 12%), 431 (8%), 381 (7%), 281 (15%) and 149 (100%); HRMS for C₂₉H₂₉N₂O₅ requires 485.2069. found 485.2076; [α]_(D) ²⁰+22.2 (c 0.94 CHCl₃).

N-Fmoc-(S)-alanyl-N-((1S)-phenyl-2-hydroxylethyl)-(R)-alanyl-(S)-alanine tert-butyl ester (71)⁴⁸

To a solution of L-alanine tert-butyl ester (66) (251 mg, 1.73 mmol, 3.0 equiv.) in anhydrous dichloromethane (25 mL) was added trimethyl aluminium (2.03 mL, 2.03 mmol, 2 M in hexane, 3.5 equiv.) under an atmosphere of nitrogen. After 15 minutes, (3R,5S)-[N4N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-2-one (37) (280 mg, 0.58 mmol) in anhydrous dichloromethane (8 mL) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction was quenched by the addition of water (7 mL) and the organic phase was then washed with saturated copper sulphate (15 mL). The aqueous phase was extracted with diethyl ether (3×20 mL) and the combined organic extracts were dried over MgSO₄. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica, eluting with petrol and diethyl ether (1:4) to furnish the title compound as fine colourless needles (226 mg, 62%); m.p. 87-88° C.; v_((max)) (KBr) 3410 (O—H), 2980 (C—H), 1727 (C═O), 1654 (C═O) cm¹; δ_(H) (250 MHz, OMSO-d), 8.35 (0.5H, d, /6.18 Hz, NH) 7.91-7.12 (13H, m, Fmoc×8 and Ph×5), 7.65 (0.5H, d, /7.14 Hz, NH), 7.55 (0.5171, d, /7.32 Hz, NH), 6.39 (0.5H, d, /7.45 Hz, NH), 5.21-5.18 (0.5H, m, PhCH), 5.03-5.01 (0.5H, m. PhCH), 4.94-4.92 (0.5H, m, NHCHCH₃), 4.80-4.78 (0.5H, m, NHCHCH₃), 4.65-4.62 (0.5H, m, NCHCH₃), 4.27-4.18 (3.5H, m, OCH₂CH, and CH₂OH×0.5), 4.09-3.93 (2.5H, m, CH₂OH×1.5 and NHCHCH₃), 3.62-3.60 (0.5H, m, NCHCH₃×1), 1.44-1.36 (13.5H, s, (CHa)₃×9, CHCH₃×4.5), 1.23 (1.5H, d, /6.63 Hz, CHCH₃), 1.13 (1.5H, d, /7.35 Hz, CHCH₃), 0.92 (1.5H, d, /7.07 Hz, CHCH₃); δ_(c) (62.5 MHz, OMSO-d) 173.4, 172.9, 171.9, 171.6, 169.7, 156.0, 144.2, 144.1, 141.1, 137.2, 129.0, 128.3, 128.0, 127.7, 127.4, 127.3, 125.7, 120.5, 81.2, 80.8, 66.1, 61.6, 55.3, 53.6, 48.6, 48.3, 47.5, 47.0, 27.9, 18.5, 18.2, 17.7, 17.1, 15.4; % (CL, NH₃), 775 (100%), 629 (M⁺, 36%), 457 (58%), 345 (27%), 231 (33%) and 178 (100%); HRMS for C₃₆H₄₃N₃O₇ requires 629.3092. found 629.3093; [α]_(D) ²⁰+14.20 (c 1.15 CHCl₃).

LDL-alanine

To a solution of N-Fmoc-(S)-alanyl-NOR)-phenyl-2-hydroxylethyl)-(R)-alanyl-(S)-alanine tert-butyl ester (235 mg, 0.37 mmol) and tert-butanol (0.12 mL, 1.20 mmol, 3.0 equiv.) in liquid ammonia (15 mL) and anhydrous tetrahydrofuran (10 mL) was added lithium (23 mg, 3.70 mmol, 10.0 equiv.) at −78° C.; under an atmosphere of nitrogen. The resulting solution was stirred until the blue colour disappeared and then allowed to warm to room temperature to evaporate off the liquid ammonia. A mixture of water (15 mL) and diethyl ether (10 mL) was added and the aqueous phase was extracted with diethyl ether (3×10 mL). Water was removed in vacuo and the crude product purified first by acidic ion exchange chromatography and then by flash column chromatography on silica, eluting with methanol and water (7:3) to furnish the title compound as fine colourless needles.

To a solution of (3S,5R)—N—[N-Fmoc-(S)-alanyl]-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (33) (1.00 g, 2.06 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (40 mL) was added 1,8-diazabicyclo[5.4.0]-undec-7-ene (0.03 mL, 0.21 mmol, 0.1 equiv.) under an atmosphere of nitrogen. The resulting solution was stirred at room temperature for 3.5 hours before N,N-diisopropylethylamine (0.40 mL, 2.26 mmol, 1.1 equiv.), N-Fmoc-L-alanine (0.76 g, 2.46 mmol, 1.2 equiv.) and bromotripyrrolidinophosphonium hexafluorophosphate (1.21 g, 2.46 mmol, 1.2 equiv.) were added. The resulting solution was stirred for another 18 hours during which time a white precipitate was formed. The mixture was filtered through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica, eluting with dichloromethane and diethyl ether (3:7) to furnish the title product as a white powder (0.96 g, 86%); m.p. 100.0-101.0° C.; v_((max))(KBr) 3311 (N—H), 2981 (C—H), 1741 (C═O), 1718 (C═O), 1647 (C═O) cm⁻¹; δ_(H) (250 MHz, DMSO-d) 7.79 (1H, /6.70 Hz, NH), 7.68-7.07 (13H, m, Fmoc×8, Ph×5), 7.32 (1H, J 7.85 Hz, NH), 5.27 (1H, br, PhCH), 5.16-4.96 (1H, m, PhCHCH₂×1), 5.16-4.96 (1H, m, PhCHCH₂×1), 4.64-4.62 (1H, m, CHCH₃), 4.55-4.51 (1H, m, CHCH₃), 4.35-4.32 (1H, m, OCH₂CH), 4.00 (2H, m, OCH₂CH), 3.89-3.80 (1H, m, CHCH₃), 0.98 (3H, d, J 13.92 Hz, CHCH₃), 0.93 (3H, br, CHCH₃), 0.62 (3H, d, /6.89 Hz, CHCH₃); δ_(c) (62.5 MHz, DMSO-d) 172.8, 171.6, 170.0, 156.0, 144.2, 141.1, 136.3, 128.9, 128.3, 128.0, 127.4, 126.8, 125.7, 120.4, 69.1, 66.0, 52.9, 50.6, 49.9, 47.0, 30.8, 20.9, 18.5, 17.8; m/z (CL, NH₃) 231 (M⁺, 40%), 230 (100%), and 228 (35%); HRMS for C₃₂H₃₃N₃O₆ requires 555.2369 found 231, 1209; [c]_(D) ²⁰ −31.3 (c 0.90 CHCl₃).

To a solution of (3S,5R)—N—[N-Fmoc-(S)-alanyl-(S)-alanine]-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one (49) (114 mg, 0.206 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (5 mL) was added 1,8-diazabicyclo[5.4.0]-undec-7-ene (0.015 mL, 0.103 mmol 0.5 equiv.) under an atmosphere of nitrogen. The resulting solution was stirred at room temperature for 5 hours before N,N-diisopropylethylamine (0.026 mL, 0.144 mmol, 0.7 equiv.), N-Fmoc-L-alanine (76 mg, 0.246 mmol, 1.2 equiv.) and bromotripyrrolidinophosphonium hexafluorophosphate (0.121 g, 0.246 mmol, 1.2 equiv.) were added. The resulting solution was stirred for another 19 hours during which time a white precipitate was formed. The mixture was filtered through a short pad of CELITE® diatomaceous earth and removal of solvent from the filtrate in vacuo yielded the crude product which was purified by flash column chromatography on silica, eluting with dichloromethane and acetone (7:3) to furnish the title product as a white powder, (89 mg, 68%); m.p. 118.0-120.0° C.; v_((max))(KBr) 3316 (N—H), 2985 (C—H), 1738 (C═O), 1708 (C═O), 1647 (C═O) cm⁻¹; δ_(H) (250 MHz, DMSO-d) 8.00 (1H, d, /7.28 Hz, NH), 7.91-7.30 (13H, m, Fmoc×8, Ph×5), 7.52 (1H, d, /7.58 Hz, NH), 5.48 (1H, br, PhCH), 5.32-5.02 (1H, m, PhCHCH₂), 4.88-4.85 (1H, m, CHCH₃), 4.74-4.69 (1H, m, CHCH₃), 4.56 (1H, br, OCH₂CH), 4.26-4.27 (2H, m, OCH₂CH), 4.03 (1H, t, /7.25 Hz, CHCH₃), 3.63-3.58 (0.5H, m, CHCH₃), 3.14-3.12 (0.5H, m, CHCH₃), 1.26-0.97 (12H, m, CHCH₃×4); δ_(c) (62.5 MHz, DMSO-d) 172.5, 172.2, 171.6, 170.0, 156.0, 144.2, 141.1, 136.5, 128.9, 128.3, 128.0, 127.4, 126.7, 125.7, 120.5, 65.9, 56.2, 53.9, 53.0, 47.9, 46.0, 42.2, 32.5, 31.0, 29.9, 18.5, 17.8, 17.1, 12.9; % (CL, NH₃); HRMS for C₃₅H₃₈N₄O₇, requires 626.2741 found; [α]_(D) ²⁰ −27.2 (c 1.04 acetone).

¹H-NMR data obtained for Tri-L-alanine made according to the invention with commercial tri-L-alanine was compared with excellent results.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

1. A compound of formula (VII):

wherein m is an integer of 1 to 50; R^(1′) is hydrogen or is independently selected from a C₁₋₁₀ branched or straight chain alkyl group, C₅₋₁₂ heteroaryl group or C₆₋₁₂ aryl group optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₂R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂; R² is independently selected from a C₁₋₁₀ branched or straight chain alkyl group, C₆₋₁₂ heteroaryl group or C₅₋₁₂ aryl group, optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, SO₂R¹², SO₂R¹², phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(NR¹³)N(R¹³)₂; R⁵ is a C₅₋₁₂ aryl, C₅₋₁₂ heteroaryl or C₁₋₆ branched or straight chain alkyl optionally substituted with OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, phenyl, imidazolyl, indolyl, hydroxyphenyl or NR¹³C(═NR¹³)N(R¹³)₂ or a linker for attachment of formula (VII) to a resin wherein the linker is selected from OR¹³, N(R¹³)₂, CO₂R¹³, SR¹³ or an alkyl group having 1 to 4 carbon atoms or a C₆-C₁₂ aryl group, wherein the alkyl and/or aryl group can be substituted with one or more of OR¹³, N(R¹³)₂, CO₂R¹³, SR¹³; R⁹ and R¹⁰ are independently hydrogen or a group as defined for R^(1′); or R⁹ and R¹⁰ can together from a 4 to 7 membered ring, optionally substituted with CO₂R¹³, OR¹³, SR¹³, N(R¹³)₂, CO₂R¹³, CON(R¹³)₂, C₁₋₁₀ alkyl or C₆₋₁₂ aryl, wherein said ring can be fully, partially or unsaturated, and wherein the ring may contain one or more heteroatoms selected from O, S or N; R¹¹ is hydrogen or an amino protecting group selected from at least one of a benzyloxycarbonyl group, a t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group or a 2-nitrophenylsulphenyl group; wherein X is O or S; R¹² is hydrogen, C₁₋₆ alkyl, C₆₋₁₂ aryl or N(R¹³)₂, wherein each occurrence of R¹³ is independently hydrogen, C₁₋₆ alkyl or C₆₋₁₂ aryl; and n is 1, 2 or
 3. 2. The compound of claim 1, wherein R^(1′) and R² are independently selected from at least one of C₁ alkyl optionally substituted with OH, SH, CO₂H, CONH₂, phenyl, imidazolyl, indolyl or hydroxyphenyl; C₂ alkyl optionally substituted with OH, CO₂H, CONH₂ or SCH₃; C₃ alkyl optionally substituted with NHC(═NH)NH₂; or C₄ alkyl optionally substituted with NH₂. 